in vitro transcription translation reaction Search Results


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    Promega in vitro transcription translation reactions
    The PcSte20 MAP kinase binds to the cell wall biosynthesis kinase PcCbk1 <t>in</t> <t>vitro</t> . To assess potential interactions of PcSte20 kinase with the putative downstream kinase PcCbk1, 35 S-labeled full-length recombinant V5 epitope-tagged PcSte20 and PcCbk1 were generated by in vitro <t>transcription-translation</t> <t>reactions,</t> purified, and allowed to interact in solution. The solutions were immunoprecipitated as described in the text, and the results of autoradiography of the precipitated products are shown. Recombinantly generated PcCbk1 revealed no immunoprecipitation with nonimmune mouse serum (lane 1). PcCbk1 was successfully precipitated with monoclonal anti- V5-agarose (lane 2). Recombinantly generated PcSte20 revealed no immunoprecipitation with nonimmune rabbit serum (lane 3). Recombinantly generated PcSte20 revealed a doublet following immunoprecipitation with an anti-PcSte20 antibody (lane 4). A mixture of PcCbk1 with PcSte20 was immunoprecipitated with anti-PcSte20 antibody, revealing precipitation of both recombinant proteins (lane 5). A control protein (luciferase) was similarly generated and revealed no precipitation with nonimmune rabbit serum (lane 6). The control luciferase protein was successfully precipitated with anti-luciferase antibody (lane 7). The PcSte20 kinase did not bind the control luciferase protein following immunoprecipitation with anti-PcSte20 antibody (lane 8).
    In Vitro Transcription Translation Reactions, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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
    Lncrna Nr 104098, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Ribobio lncrna snhg7
    <t>LncRNA-</t> <t>SNHG7</t> promotes proliferation of breast cancer cells. (A, B) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus. LncRNA- SNHG7 level was assessed by qRT-PCR, and then cells were subjected to cell proliferation analysis. (C) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus and then subjected to cell viability assays. (D, E) MCF-7 cells were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus. LncRNA- SNHG7 level was assessed by qRT-PCR, and then the cells were subjected to cell proliferation analysis. (F) MCF-7 cells were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus, and then subjected to cell viability assays. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; qRT-PCR = quantitative real-time polymerase chain reaction; sh = short hairpin. * p
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    97
    Promega in vitro transcription translation reaction
    <t>LncRNA-</t> <t>SNHG7</t> promotes proliferation of breast cancer cells. (A, B) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus. LncRNA- SNHG7 level was assessed by qRT-PCR, and then cells were subjected to cell proliferation analysis. (C) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus and then subjected to cell viability assays. (D, E) MCF-7 cells were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus. LncRNA- SNHG7 level was assessed by qRT-PCR, and then the cells were subjected to cell proliferation analysis. (F) MCF-7 cells were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus, and then subjected to cell viability assays. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; qRT-PCR = quantitative real-time polymerase chain reaction; sh = short hairpin. * p
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    Image Search Results


    The PcSte20 MAP kinase binds to the cell wall biosynthesis kinase PcCbk1 in vitro . To assess potential interactions of PcSte20 kinase with the putative downstream kinase PcCbk1, 35 S-labeled full-length recombinant V5 epitope-tagged PcSte20 and PcCbk1 were generated by in vitro transcription-translation reactions, purified, and allowed to interact in solution. The solutions were immunoprecipitated as described in the text, and the results of autoradiography of the precipitated products are shown. Recombinantly generated PcCbk1 revealed no immunoprecipitation with nonimmune mouse serum (lane 1). PcCbk1 was successfully precipitated with monoclonal anti- V5-agarose (lane 2). Recombinantly generated PcSte20 revealed no immunoprecipitation with nonimmune rabbit serum (lane 3). Recombinantly generated PcSte20 revealed a doublet following immunoprecipitation with an anti-PcSte20 antibody (lane 4). A mixture of PcCbk1 with PcSte20 was immunoprecipitated with anti-PcSte20 antibody, revealing precipitation of both recombinant proteins (lane 5). A control protein (luciferase) was similarly generated and revealed no precipitation with nonimmune rabbit serum (lane 6). The control luciferase protein was successfully precipitated with anti-luciferase antibody (lane 7). The PcSte20 kinase did not bind the control luciferase protein following immunoprecipitation with anti-PcSte20 antibody (lane 8).

    Journal: Infection and Immunity

    Article Title: Pneumocystis carinii Interactions with Lung Epithelial Cells and Matrix Proteins Induce Expression and Activity of the PcSte20 Kinase with Subsequent Phosphorylation of the Downstream Cell Wall Biosynthesis Kinase PcCbk1 ▿

    doi: 10.1128/IAI.05066-11

    Figure Lengend Snippet: The PcSte20 MAP kinase binds to the cell wall biosynthesis kinase PcCbk1 in vitro . To assess potential interactions of PcSte20 kinase with the putative downstream kinase PcCbk1, 35 S-labeled full-length recombinant V5 epitope-tagged PcSte20 and PcCbk1 were generated by in vitro transcription-translation reactions, purified, and allowed to interact in solution. The solutions were immunoprecipitated as described in the text, and the results of autoradiography of the precipitated products are shown. Recombinantly generated PcCbk1 revealed no immunoprecipitation with nonimmune mouse serum (lane 1). PcCbk1 was successfully precipitated with monoclonal anti- V5-agarose (lane 2). Recombinantly generated PcSte20 revealed no immunoprecipitation with nonimmune rabbit serum (lane 3). Recombinantly generated PcSte20 revealed a doublet following immunoprecipitation with an anti-PcSte20 antibody (lane 4). A mixture of PcCbk1 with PcSte20 was immunoprecipitated with anti-PcSte20 antibody, revealing precipitation of both recombinant proteins (lane 5). A control protein (luciferase) was similarly generated and revealed no precipitation with nonimmune rabbit serum (lane 6). The control luciferase protein was successfully precipitated with anti-luciferase antibody (lane 7). The PcSte20 kinase did not bind the control luciferase protein following immunoprecipitation with anti-PcSte20 antibody (lane 8).

    Article Snippet: All in vitro transcription-translation reactions (TNT reactions) were performed in 50.0-μl volumes using 1.0 μg of the uncut plasmid along with the T7 coupled reticulocyte lysate system (Promega Inc., Madison, WI) in the presence of 40.0 μl of [35 S]methionine (1,000 Ci/mmol) at 30°C for 90.0 min.

    Techniques: In Vitro, Labeling, Recombinant, Generated, Purification, Immunoprecipitation, Autoradiography, Luciferase

    A specific region of PcCbk1 binds to the upstream PcSte20 kinase. To further determine the site of PcCbk1's interaction with PcSte20, 35 S-labeled full-length recombinant V5 epitope-tagged PcSte20 and PcCbk1 and three recombinant fragments of PcCbk1 were generated by in vitro transcription-translation reactions, purified, and allowed to interact in solution. The solutions were immunoprecipitated as described in the text, and the results of autoradiography of the precipitated products are shown. Full-length PcCbk1 was immunoprecipitated with monoclonal anti-V5 agarose (lane 1). Full-length PcSte20 immunoprecipitated with PcSte20 antibody revealed a doublet of the appropriate molecular mass (lane 2). The PcCbk1-1-507 fragment was generated and precipitated with anti-V5 agarose (lane 3). The PcCbk1-508-1014 fragment was generated and precipitated with anti-V5 agarose (lane 4). The PcCbk1-1015-1524 fragment was generated and precipitated with anti-V5 agarose (lane 5). Recombinant full-length PcCbk1 and PcSte20 were incubated together and immunoprecipitated with anti-PcSte20 antibody, demonstrating coimmunoprecipitation of the two kinases (lane 6). PcCbk1-1-507 (filled arrowheads) and PcSte20 (open arrowheads) were coincubated and immunoprecipitated with antibody to PcSte20, revealing the specific association of this fragment of the molecule (lane 7). In contrast, neither PcCbk1-508-1014 nor PcCbk1-1015-1524 associated with PcSte20 following immunoprecipitation with PcSte20 antibody (lanes 8 and 9).

    Journal: Infection and Immunity

    Article Title: Pneumocystis carinii Interactions with Lung Epithelial Cells and Matrix Proteins Induce Expression and Activity of the PcSte20 Kinase with Subsequent Phosphorylation of the Downstream Cell Wall Biosynthesis Kinase PcCbk1 ▿

    doi: 10.1128/IAI.05066-11

    Figure Lengend Snippet: A specific region of PcCbk1 binds to the upstream PcSte20 kinase. To further determine the site of PcCbk1's interaction with PcSte20, 35 S-labeled full-length recombinant V5 epitope-tagged PcSte20 and PcCbk1 and three recombinant fragments of PcCbk1 were generated by in vitro transcription-translation reactions, purified, and allowed to interact in solution. The solutions were immunoprecipitated as described in the text, and the results of autoradiography of the precipitated products are shown. Full-length PcCbk1 was immunoprecipitated with monoclonal anti-V5 agarose (lane 1). Full-length PcSte20 immunoprecipitated with PcSte20 antibody revealed a doublet of the appropriate molecular mass (lane 2). The PcCbk1-1-507 fragment was generated and precipitated with anti-V5 agarose (lane 3). The PcCbk1-508-1014 fragment was generated and precipitated with anti-V5 agarose (lane 4). The PcCbk1-1015-1524 fragment was generated and precipitated with anti-V5 agarose (lane 5). Recombinant full-length PcCbk1 and PcSte20 were incubated together and immunoprecipitated with anti-PcSte20 antibody, demonstrating coimmunoprecipitation of the two kinases (lane 6). PcCbk1-1-507 (filled arrowheads) and PcSte20 (open arrowheads) were coincubated and immunoprecipitated with antibody to PcSte20, revealing the specific association of this fragment of the molecule (lane 7). In contrast, neither PcCbk1-508-1014 nor PcCbk1-1015-1524 associated with PcSte20 following immunoprecipitation with PcSte20 antibody (lanes 8 and 9).

    Article Snippet: All in vitro transcription-translation reactions (TNT reactions) were performed in 50.0-μl volumes using 1.0 μg of the uncut plasmid along with the T7 coupled reticulocyte lysate system (Promega Inc., Madison, WI) in the presence of 40.0 μl of [35 S]methionine (1,000 Ci/mmol) at 30°C for 90.0 min.

    Techniques: Labeling, Recombinant, Generated, In Vitro, Purification, Immunoprecipitation, Autoradiography, Incubation

    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: In addition, qPCR results revealed that ATPR induced expression of lncRNA NR-104098 in a time- and concentration-dependent manner ( ).

    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: In addition, qPCR results revealed that ATPR induced expression of lncRNA NR-104098 in a time- and concentration-dependent manner ( ).

    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: In addition, qPCR results revealed that ATPR induced expression of lncRNA NR-104098 in a time- and concentration-dependent manner ( ).

    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: In addition, qPCR results revealed that ATPR induced expression of lncRNA NR-104098 in a time- and concentration-dependent manner ( ).

    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: In addition, qPCR results revealed that ATPR induced expression of lncRNA NR-104098 in a time- and concentration-dependent manner ( ).

    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: In addition, qPCR results revealed that ATPR induced expression of lncRNA NR-104098 in a time- and concentration-dependent manner ( ).

    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: In addition, qPCR results revealed that ATPR induced expression of lncRNA NR-104098 in a time- and concentration-dependent manner ( ).

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

    LncRNA- SNHG7 promotes proliferation of breast cancer cells. (A, B) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus. LncRNA- SNHG7 level was assessed by qRT-PCR, and then cells were subjected to cell proliferation analysis. (C) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus and then subjected to cell viability assays. (D, E) MCF-7 cells were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus. LncRNA- SNHG7 level was assessed by qRT-PCR, and then the cells were subjected to cell proliferation analysis. (F) MCF-7 cells were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus, and then subjected to cell viability assays. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; qRT-PCR = quantitative real-time polymerase chain reaction; sh = short hairpin. * p

    Journal: Journal of Breast Cancer

    Article Title: c-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer

    doi: 10.4048/jbc.2019.22.e54

    Figure Lengend Snippet: LncRNA- SNHG7 promotes proliferation of breast cancer cells. (A, B) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus. LncRNA- SNHG7 level was assessed by qRT-PCR, and then cells were subjected to cell proliferation analysis. (C) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus and then subjected to cell viability assays. (D, E) MCF-7 cells were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus. LncRNA- SNHG7 level was assessed by qRT-PCR, and then the cells were subjected to cell proliferation analysis. (F) MCF-7 cells were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus, and then subjected to cell viability assays. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; qRT-PCR = quantitative real-time polymerase chain reaction; sh = short hairpin. * p

    Article Snippet: The lncRNA-SNHG7 and miR-34a-5p levels were analyzed by quantitative RT-PCR (qRT-PCR).

    Techniques: Transduction, Quantitative RT-PCR, Standard Deviation, Real-time Polymerase Chain Reaction

    LncRNA- SNHG7 is a direct transcriptional target of c-Myc. (A, B) MCF-7 and MDA-MB-231 cells were transduced with sh-ctrl or sh-c-Myc lentivirus, pCDH or pCDH-c-Myc lentivirus. Western blot analysis of c-Myc and β-actin. LncRNA- SNHG7 level was assessed by quantitative RT-PCR. (C) Schematic illustration of the consensus c-Myc binding sites in lncRNA- SNHG7 gene promoter. The wild-type and mutant binding sites are shown in the open boxes. The corresponding pGL3-based luciferase reporter constructs generated are shown. (D, E) MCF-7 were transduced with sh-ctrl or sh-c-Myc lentivirus, pCDH or pCDH-c-Myc lentivirus followed by transfection with the indicated pGL3-based luciferase reporter constructs. The reporter activity was measured 24 hours post-transfection, and plotted after normalizing with the Renilla luciferase activity. Western blot analysis of c-Myc and β-actin. (F) MCF-7 cells lysates were analyzed by ChIP assay using anti-c-Myc or IgG rabbit antibody. The ChIP products were amplified by semi-quantitative RT-PCR. (G) Correlation analyses conducted between c-Myc and lncRNA- SNHG7 in breast cancers (n = 50 biologically independent samples). Pearson correlation coefficients ( r ) and p -values. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; RT-PCR = real-time polymerase chain reaction; NS = not significant; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IgG = immunoglobulin G. * p

    Journal: Journal of Breast Cancer

    Article Title: c-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer

    doi: 10.4048/jbc.2019.22.e54

    Figure Lengend Snippet: LncRNA- SNHG7 is a direct transcriptional target of c-Myc. (A, B) MCF-7 and MDA-MB-231 cells were transduced with sh-ctrl or sh-c-Myc lentivirus, pCDH or pCDH-c-Myc lentivirus. Western blot analysis of c-Myc and β-actin. LncRNA- SNHG7 level was assessed by quantitative RT-PCR. (C) Schematic illustration of the consensus c-Myc binding sites in lncRNA- SNHG7 gene promoter. The wild-type and mutant binding sites are shown in the open boxes. The corresponding pGL3-based luciferase reporter constructs generated are shown. (D, E) MCF-7 were transduced with sh-ctrl or sh-c-Myc lentivirus, pCDH or pCDH-c-Myc lentivirus followed by transfection with the indicated pGL3-based luciferase reporter constructs. The reporter activity was measured 24 hours post-transfection, and plotted after normalizing with the Renilla luciferase activity. Western blot analysis of c-Myc and β-actin. (F) MCF-7 cells lysates were analyzed by ChIP assay using anti-c-Myc or IgG rabbit antibody. The ChIP products were amplified by semi-quantitative RT-PCR. (G) Correlation analyses conducted between c-Myc and lncRNA- SNHG7 in breast cancers (n = 50 biologically independent samples). Pearson correlation coefficients ( r ) and p -values. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; RT-PCR = real-time polymerase chain reaction; NS = not significant; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IgG = immunoglobulin G. * p

    Article Snippet: The lncRNA-SNHG7 and miR-34a-5p levels were analyzed by quantitative RT-PCR (qRT-PCR).

    Techniques: Multiple Displacement Amplification, Transduction, Western Blot, Quantitative RT-PCR, Binding Assay, Mutagenesis, Luciferase, Construct, Generated, Transfection, Activity Assay, Chromatin Immunoprecipitation, Amplification, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Elevated expression of lncRNA- SNHG7 in breast cancer. (A) qRT-PCR for the abundance of lncRNA- SNHG7 in 30 pairs of breast cancer samples. (B) The correlation between overall survival and lncRNA- SNHG7 level in breast cancer obtained by TANRIC. (C) qRT-PCR for the abundance of lncRNA- SNHG7 in normal breast cells and various breast cancer cell lines. (D) Western-blot analysis for PARP and β-actin in the nuclear and cytoplasmic fractions, respectively, in MCF-7 cells. Data shown represent 3 independent experiments. (E) Nuclear and cytoplasmic fractions of MCF-7 cells were subjected to qRT-PCR analysis. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; qRT-PCR = quantitative real-time polymerase chain reaction; PARP = poly (ADP-ribose) polymerase. * p

    Journal: Journal of Breast Cancer

    Article Title: c-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer

    doi: 10.4048/jbc.2019.22.e54

    Figure Lengend Snippet: Elevated expression of lncRNA- SNHG7 in breast cancer. (A) qRT-PCR for the abundance of lncRNA- SNHG7 in 30 pairs of breast cancer samples. (B) The correlation between overall survival and lncRNA- SNHG7 level in breast cancer obtained by TANRIC. (C) qRT-PCR for the abundance of lncRNA- SNHG7 in normal breast cells and various breast cancer cell lines. (D) Western-blot analysis for PARP and β-actin in the nuclear and cytoplasmic fractions, respectively, in MCF-7 cells. Data shown represent 3 independent experiments. (E) Nuclear and cytoplasmic fractions of MCF-7 cells were subjected to qRT-PCR analysis. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; qRT-PCR = quantitative real-time polymerase chain reaction; PARP = poly (ADP-ribose) polymerase. * p

    Article Snippet: The lncRNA-SNHG7 and miR-34a-5p levels were analyzed by quantitative RT-PCR (qRT-PCR).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction

    Knocking down lncRNA- SNHG7 inhibits glycolysis in breast cancer cells. (A) MCF-7 cells expressing either control shRNA or lncRNA- SNHG7 shRNA were cultured for 24 hours. Acidification of the culture medium was evaluated by visually inspecting the color of the medium. (B) MCF-7 cells expressing either control shRNA or lncRNA- SNHG7 shRNA were cultured for 24 hours. Levels of lactate in the culture medium were then measured and normalized to cell number. (C, D) ECAR was measured in MCF-7 cells expressing either control shRNA or lncRNA- SNHG7 shRNA by Seahorse XF assays. (E, F) Western blotting and qRT-PCR analysis of glycolysis enzymes in MCF-7 cells expressing either control shRNA or lncRNA- SNHG7 shRNA. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; ECAR = extracellular acidification rate; qRT-PCR = quantitative real-time polymerase chain reaction. * p

    Journal: Journal of Breast Cancer

    Article Title: c-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer

    doi: 10.4048/jbc.2019.22.e54

    Figure Lengend Snippet: Knocking down lncRNA- SNHG7 inhibits glycolysis in breast cancer cells. (A) MCF-7 cells expressing either control shRNA or lncRNA- SNHG7 shRNA were cultured for 24 hours. Acidification of the culture medium was evaluated by visually inspecting the color of the medium. (B) MCF-7 cells expressing either control shRNA or lncRNA- SNHG7 shRNA were cultured for 24 hours. Levels of lactate in the culture medium were then measured and normalized to cell number. (C, D) ECAR was measured in MCF-7 cells expressing either control shRNA or lncRNA- SNHG7 shRNA by Seahorse XF assays. (E, F) Western blotting and qRT-PCR analysis of glycolysis enzymes in MCF-7 cells expressing either control shRNA or lncRNA- SNHG7 shRNA. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; ECAR = extracellular acidification rate; qRT-PCR = quantitative real-time polymerase chain reaction. * p

    Article Snippet: The lncRNA-SNHG7 and miR-34a-5p levels were analyzed by quantitative RT-PCR (qRT-PCR).

    Techniques: Expressing, shRNA, Cell Culture, Western Blot, Quantitative RT-PCR, Standard Deviation, Real-time Polymerase Chain Reaction

    LncRNA- SNHG7 acts as a target of miR-34a-5p to increase LDHA level. (A) Illustration of the base pairing between miR-34a-5p and lncRNA- SNHG7 . The base pairing between miR-34a-5p and LDHA 3′UTR is also shown. (B) Lysates from MCF-7 cells were incubated with in vitro -synthesized biotin-labeled sense or antisense DNA probes against lncRNA- SNHG7 for biotin pull-down assay, followed by qRT-PCR analysis to examine miR-34a-5p and lncRNA- SNHG7 level. (C) MCF-7 were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus followed by miR-34a-5p inhibitor or negative control transfection. Western blot analysis of LDHA and β-actin. (D) MCF-7 were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus followed by miR-34a-5p mimics or negative control transfection. Western blot analysis of LDHA and β-actin. (E, F) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus followed by miR-34a-5p inhibitor or negative control transfection. ECAR was measured by Seahorse XF assays. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; ECAR = extracellular acidification rate; qRT-PCR = quantitative real-time polymerase chain reaction; ns = not significant; LDHA = lactate dehydrogenase A; 3′UTR = 3′ untranslated region. * p

    Journal: Journal of Breast Cancer

    Article Title: c-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer

    doi: 10.4048/jbc.2019.22.e54

    Figure Lengend Snippet: LncRNA- SNHG7 acts as a target of miR-34a-5p to increase LDHA level. (A) Illustration of the base pairing between miR-34a-5p and lncRNA- SNHG7 . The base pairing between miR-34a-5p and LDHA 3′UTR is also shown. (B) Lysates from MCF-7 cells were incubated with in vitro -synthesized biotin-labeled sense or antisense DNA probes against lncRNA- SNHG7 for biotin pull-down assay, followed by qRT-PCR analysis to examine miR-34a-5p and lncRNA- SNHG7 level. (C) MCF-7 were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus followed by miR-34a-5p inhibitor or negative control transfection. Western blot analysis of LDHA and β-actin. (D) MCF-7 were transduced with pCDH or pCDH-lncRNA- SNHG7 lentivirus followed by miR-34a-5p mimics or negative control transfection. Western blot analysis of LDHA and β-actin. (E, F) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA- SNHG7 lentivirus followed by miR-34a-5p inhibitor or negative control transfection. ECAR was measured by Seahorse XF assays. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; ECAR = extracellular acidification rate; qRT-PCR = quantitative real-time polymerase chain reaction; ns = not significant; LDHA = lactate dehydrogenase A; 3′UTR = 3′ untranslated region. * p

    Article Snippet: The lncRNA-SNHG7 and miR-34a-5p levels were analyzed by quantitative RT-PCR (qRT-PCR).

    Techniques: Incubation, In Vitro, Synthesized, Labeling, Pull Down Assay, Quantitative RT-PCR, Transduction, Negative Control, Transfection, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction