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GenePharma Company lncrna nr 104098
<t>lncRNA</t> <t>NR-104098</t> downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
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1) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

2) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

3) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

4) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

5) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

6) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

7) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

8) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

9) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

10) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

11) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

12) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

13) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

14) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

15) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

16) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

17) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

18) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

19) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

20) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

21) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

22) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

23) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

24) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

25) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

26) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

27) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

28) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

29) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

30) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

31) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

32) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

33) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

34) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

35) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

36) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

37) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

38) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

39) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

40) Product Images from "LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1"

Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00142

lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p
Figure Legend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.
Figure Legend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

Techniques Used: Over Expression, Binding Assay, Expressing, Inhibition

Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
Figure Legend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

Techniques Used: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay

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    GenePharma Company lncrna nr 104098
    <t>lncRNA</t> <t>NR-104098</t> downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p
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    lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

    doi: 10.3389/fcell.2020.00142

    Figure Lengend Snippet: lncRNA NR-104098 downregulated transcriptional expression of EZH2 through recruitment of E2F1 in AML cells. (A) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 antibodies. (B) AML cells transfected with E2F1 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F1 protein levels were detected by Western-blot assay. (C) E2F1 siRNAs (E2F1-1, 2) or the control siRNA were transfected into AML cells for 48 h, E2F1 and EZH2 mRNA levels were then assessed by RT-qPCR. (D) RNA immunoprecipitations were performed in AML cells, and the relative quantities of lncRNA NR-104098 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F1 represented for the groups coprecipitation with IgG protein and anti- E2F1 antibody respectively. (E) Total proteins were extracted from NB4 and THP-1 cells, and then lncRNA NR-104098 pull-down assay was performed. The E2F1 protein levels were evaluated by Western-blot. LncRNA NR-104098 probe represented the biotin-labeled lncRNA NR-104098 probe group and control stood for the oligo probe group. NB4 and THP-1 cells with pEGFP-C3-NR-104098 or the empty vector was harvested, and expression levels of E2F1 protein (F) and E2F1 mRNA (G) were detected. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Article Snippet: Knocking down lncRNA NR-104098 can also reverse the downregulation of EZH2 mRNA transcription activity induced by ATPR ( ).

    Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Pull Down Assay, Labeling

    Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

    doi: 10.3389/fcell.2020.00142

    Figure Lengend Snippet: Upregulation of lncRNA NR-104098 suppressed proliferation and induced differentiation of AML cells in vivo . Four-week-old NCG mice were randomly divided into two groups, and NB4 cells (1 × 10 6 ) with pEGFP-C3-NR-104098 or empty vector stable transfection were injected s.c. respectively. 8 weeks after that, the mice were sacrificed and the tumors were removed, photographed (A) , and weighed (B,C) . (D) Then, protein was extracted from the tumor tissues, and expressions of EZH2 protein was detected by Western blot. (E) Immunohistochemistry analysis of EZH2 was obtained from tumors. (F) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. (G) Western blot analysis of CD11b and CD14 in tumor tissues of pEGFP-C3-NR-104098 or empty vector groups. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Article Snippet: Knocking down lncRNA NR-104098 can also reverse the downregulation of EZH2 mRNA transcription activity induced by ATPR ( ).

    Techniques: In Vivo, Mouse Assay, Plasmid Preparation, Stable Transfection, Injection, Western Blot, Immunohistochemistry

    LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

    doi: 10.3389/fcell.2020.00142

    Figure Lengend Snippet: LncRNA NR-104098 overexpression suppressed proliferation and induced differentiation of AML cells in vitro . (A) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells. 48 h later, overexpression efficient of lncRNA NR-104098 was evaluated by RT-qPCR. (B) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 24, 48, and 72 h respectively, and cell viabilities were assessed by CCK8 assay. (C) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and ki67 protein expression was detected by immunofluorescence. (D) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and the cell cycle were analyzed by flow cytometry. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. (F) pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells for 48 h, and cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after pEGFP-C3-NR-104098 or the empty vector was transfected into AML cells. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Article Snippet: Knocking down lncRNA NR-104098 can also reverse the downregulation of EZH2 mRNA transcription activity induced by ATPR ( ).

    Techniques: Over Expression, In Vitro, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, CCK-8 Assay, Expressing, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

    ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

    doi: 10.3389/fcell.2020.00142

    Figure Lengend Snippet: ATPR Induced the Expression of lncRNA NR-104098. (A) NB4 cells were treated with ATPR (10 –6 M) at different time points (0, 24, 48, and 72 h). NB4 cells were treated with an ATPR concentration gradient (0–10 –5 M) for 72 h. Then, the mRNA expression of lncRNA NR-104098 were assessed by RT-PCR. (B) RT-PCR analysis of lncRNA NR-104098 expression in AML cell lines and SC cells. (C,D) lncRNA NR-104098 distribution detected by FISH. Values were presented as mean ± SD of three independent experiments. * p

    Article Snippet: Knocking down lncRNA NR-104098 can also reverse the downregulation of EZH2 mRNA transcription activity induced by ATPR ( ).

    Techniques: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

    Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

    doi: 10.3389/fcell.2020.00142

    Figure Lengend Snippet: Knockdown of lncRNA NR-104098 inhibited ATPR-suppressed proliferation and induced differentiation of AML cells in vitro . (A) The expression levels of lncRNA NR-104098 in NB4 and THP-1 cells were detected by RT-qPCR assay, with transfection of lncRNA NR-104098 shRNA or the NC shRNA for 48 h. (B) AML cells were treated with ATPR or Solvent. 72 h later, cells were collected and cell viabilities were evaluated by CCK8 assay. (C) Meanwhile, ki67 protein expression detected by immunofluorescence. (D) Cell cycle were analyzed by flow cytometry analyzed. (E) Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 protein levels in AML cells after treated. (F) Cell differentiation were analyzed by flow cytometry. (G) Western blot analysis of CD11b and CD14 protein levels in AML cells after treated. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Article Snippet: Knocking down lncRNA NR-104098 can also reverse the downregulation of EZH2 mRNA transcription activity induced by ATPR ( ).

    Techniques: In Vitro, Expressing, Quantitative RT-PCR, Transfection, shRNA, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Western Blot, Cell Differentiation

    A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

    doi: 10.3389/fcell.2020.00142

    Figure Lengend Snippet: A hypothetical working model of the role of lncRNA NR-104098 in AML proliferation and differentiation. Overexpression of lncRNA NR-104098 effectively increases the binding of E2F1 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that inhibits AML cell proliferation and induces differentiation. Meanwhile, an ATRA derivative ATPR could inhibit AML cell proliferation and induce differentiation though regulating the expression of lncRNA NR-104098. The arrows refers to the role of promotion, and the symbol of “T” refers to the role of inhibition.

    Article Snippet: Knocking down lncRNA NR-104098 can also reverse the downregulation of EZH2 mRNA transcription activity induced by ATPR ( ).

    Techniques: Over Expression, Binding Assay, Expressing, Inhibition

    Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: LncRNA NR-104098 Inhibits AML Proliferation and Induces Differentiation Through Repressing EZH2 Transcription by Interacting With E2F1

    doi: 10.3389/fcell.2020.00142

    Figure Lengend Snippet: Upregulation of lncRNA NR-104098 inhibited EZH2 transcription in AML cells. (A) Microarray analyses results showed that lncRNA NR-104098 may regulate the oncogene EZH2. (B) GO process analysis results of lncRNA NR-104098 regulates cell cycle. (C) pEGFP-C3-NR-104098 or the empty vector was stably transfected into AML cells, and EZH2 protein levels were detected by Western-blot assay. (D) lncRNA NR-104098 shRNA or the NC shRNA was stably transfected into AML cells, the ATPR or Solvent was treated for 72 h, and the Western-blot assay indicated that lncRNA NR-104098 shRNA evidently inhibited ATPR-induced downregulation of EZH2 protein expression. (E) Either AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. (F) Either AML cells stably transfected with lncRNA NR-104098 shRNA or NC shRNA were treated with ATPR or Solvent for 72 h, RT-qPCR showed that lncRNA NR-104098 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by ATPR. (G) Either EZH2 transcriptional activities of AML cells stably transfected with pEGFP-C3-NR-104098 or the empty vector were assessed by luciferase reporter assay. (H) The cells were then treated with ATPR or Solvent. 72 h later, EZH2 transcriptional activities were analyzed, and the activity of firefly luciferase was normalized by luciferase reporter assay. β-Actin served as a loading control. Data are presented as the mean ± SD of three independent experiments. ∗ p

    Article Snippet: Knocking down lncRNA NR-104098 can also reverse the downregulation of EZH2 mRNA transcription activity induced by ATPR ( ).

    Techniques: Microarray, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, shRNA, Expressing, Quantitative RT-PCR, Inhibition, Luciferase, Reporter Assay, Activity Assay