Structured Review

Roche biotin rna labeling mix
AVAN direct binds to TRIM25 and enhances the antivirus immune response (A) <t>RNA</t> pull-down of AVAN -associated proteins using biotinylated AVAN or <t>antisense</t> probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. (B) Pull-down western blot showing that AVAN can bind directly to TRIM25. (C) ChIRP followed by western blot show that AVAN can bind to TRIM25. (D and E) Exogenous (D) and endogenous (E) RIP of TRIM25 in BJ501 infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR. (F) AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD or Flag-Ring. (G) Truncated AVAN pull-down, truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pulldown. (H and I) TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN s or siRNAs, followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. (J and K) Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVAN s or siRNAs. Anti-RIG-I antibody was used for immunoprecipitated. (L) Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN , HA-Ub and Flag-tagged RIG-I. (M and N) IFN-alpha (M) and IFN-beta (N) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI=1) at 24h post-infection, and then individually knock down RIG-I or TRIM25, analyzed by qRT-PCR (n=3; means ± SEM; *p
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1) Product Images from "Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a"

Article Title: Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a

Journal: bioRxiv

doi: 10.1101/623132

AVAN direct binds to TRIM25 and enhances the antivirus immune response (A) RNA pull-down of AVAN -associated proteins using biotinylated AVAN or antisense probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. (B) Pull-down western blot showing that AVAN can bind directly to TRIM25. (C) ChIRP followed by western blot show that AVAN can bind to TRIM25. (D and E) Exogenous (D) and endogenous (E) RIP of TRIM25 in BJ501 infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR. (F) AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD or Flag-Ring. (G) Truncated AVAN pull-down, truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pulldown. (H and I) TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN s or siRNAs, followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. (J and K) Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVAN s or siRNAs. Anti-RIG-I antibody was used for immunoprecipitated. (L) Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN , HA-Ub and Flag-tagged RIG-I. (M and N) IFN-alpha (M) and IFN-beta (N) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI=1) at 24h post-infection, and then individually knock down RIG-I or TRIM25, analyzed by qRT-PCR (n=3; means ± SEM; *p
Figure Legend Snippet: AVAN direct binds to TRIM25 and enhances the antivirus immune response (A) RNA pull-down of AVAN -associated proteins using biotinylated AVAN or antisense probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. (B) Pull-down western blot showing that AVAN can bind directly to TRIM25. (C) ChIRP followed by western blot show that AVAN can bind to TRIM25. (D and E) Exogenous (D) and endogenous (E) RIP of TRIM25 in BJ501 infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR. (F) AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD or Flag-Ring. (G) Truncated AVAN pull-down, truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pulldown. (H and I) TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN s or siRNAs, followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. (J and K) Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVAN s or siRNAs. Anti-RIG-I antibody was used for immunoprecipitated. (L) Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN , HA-Ub and Flag-tagged RIG-I. (M and N) IFN-alpha (M) and IFN-beta (N) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI=1) at 24h post-infection, and then individually knock down RIG-I or TRIM25, analyzed by qRT-PCR (n=3; means ± SEM; *p

Techniques Used: Isolation, SDS Page, Silver Staining, Western Blot, Infection, Quantitative RT-PCR, Transfection, In Vitro, Incubation, Immunoprecipitation, Expressing

2) Product Images from "Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer"

Article Title: Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer

Journal: American Journal of Cancer Research

doi:

Association of AFAP1-AS1 and Polycomb Repressive Complex 2. (A) RIP enrichment was determined as RNA associated with EZH2 IP relative to an input control. (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate (IP). (C) Biotinylated AFAP1-AS1 was incubated with nuclear extracts (SW480 and HCT116 cells), targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and AFAP1-AS1 (n = 3). (D, E) RNAs corresponding to different fragments of AFAP1-AS1 were treated as in (C), and associated EZH2 was detected by western blotting (n = 3). Error bars ± SD. *, P
Figure Legend Snippet: Association of AFAP1-AS1 and Polycomb Repressive Complex 2. (A) RIP enrichment was determined as RNA associated with EZH2 IP relative to an input control. (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate (IP). (C) Biotinylated AFAP1-AS1 was incubated with nuclear extracts (SW480 and HCT116 cells), targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and AFAP1-AS1 (n = 3). (D, E) RNAs corresponding to different fragments of AFAP1-AS1 were treated as in (C), and associated EZH2 was detected by western blotting (n = 3). Error bars ± SD. *, P

Techniques Used: Incubation, Western Blot

3) Product Images from "LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3"

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0873-2

DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P
Figure Legend Snippet: DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P

Techniques Used: Expressing, Quantitative RT-PCR, Labeling, Incubation, SDS Page, Electrophoresis, Silver Staining, Western Blot, Isolation, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Over Expression

4) Product Images from "Role of the ATPase/helicase maleless (MLE) in the assembly, targeting, spreading and function of the male-specific lethal (MSL) complex of Drosophila"

Article Title: Role of the ATPase/helicase maleless (MLE) in the assembly, targeting, spreading and function of the male-specific lethal (MSL) complex of Drosophila

Journal: Epigenetics & Chromatin

doi: 10.1186/1756-8935-4-6

Summary of the known interactions of the male-specific lethal (MSL) complex subunits . MSL1 and MSL2 form a DNA-binding module, the specificity of which requires the association of MSL2 with roX RNA. The association of MSL1 and MSL3 with MOF enhances its histone acetyltransferase activity and specificity. MLE and MSL2 interact and contribute to MOF activity. Maleless (MLE) is responsible for the incorporation of roX RNA in the complex and requires roX RNA to associate with the complex. The roX RNA is needed for full histone acetylation. Please see the discussion section for details of these associations.
Figure Legend Snippet: Summary of the known interactions of the male-specific lethal (MSL) complex subunits . MSL1 and MSL2 form a DNA-binding module, the specificity of which requires the association of MSL2 with roX RNA. The association of MSL1 and MSL3 with MOF enhances its histone acetyltransferase activity and specificity. MLE and MSL2 interact and contribute to MOF activity. Maleless (MLE) is responsible for the incorporation of roX RNA in the complex and requires roX RNA to associate with the complex. The roX RNA is needed for full histone acetylation. Please see the discussion section for details of these associations.

Techniques Used: Binding Assay, Activity Assay

5) Product Images from "Long noncoding RNA GAPLINC promotes invasion in colorectal cancer by targeting SNAI2 through binding with PSF and NONO"

Article Title: Long noncoding RNA GAPLINC promotes invasion in colorectal cancer by targeting SNAI2 through binding with PSF and NONO

Journal: Oncotarget

doi: 10.18632/oncotarget.9741

GAPLINC bound to PSF and NONO A. RNA pull-down was performed using a GAPLINC template and RNA-bound protein separated by SDS-PAGE in HCT116. The protein bands were excised and detected by mass spectrometry analysis. B. PSF and NONO were detected by the Western blotting assay in the samples pulled down by GAPLINC. C. RIP analyses were performed using antibodies against PSF and NONO, with IgG as a negative control in HCT116. The enrichment of the GAPLINC was detected using RT-PCR and normalized to the input. D. The Transwell assay was performed to assess the invasion ability of HCT116-GAPLINC-shRNA cells transiently transfected with empty vector, PSF-siRNA, and NONO-siRNA. E. Western blotting analysis showed that the protein levels of PSF, NONO, SNAI2, and GAPDH were detected in HCT116 empty vector, GAPLINC-siRNA, GAPLINC-pcDNA3.1, PSF-siRNA, and NONO-siRNA cells. Data represent mean ± standard deviation from three independent experiments. * P
Figure Legend Snippet: GAPLINC bound to PSF and NONO A. RNA pull-down was performed using a GAPLINC template and RNA-bound protein separated by SDS-PAGE in HCT116. The protein bands were excised and detected by mass spectrometry analysis. B. PSF and NONO were detected by the Western blotting assay in the samples pulled down by GAPLINC. C. RIP analyses were performed using antibodies against PSF and NONO, with IgG as a negative control in HCT116. The enrichment of the GAPLINC was detected using RT-PCR and normalized to the input. D. The Transwell assay was performed to assess the invasion ability of HCT116-GAPLINC-shRNA cells transiently transfected with empty vector, PSF-siRNA, and NONO-siRNA. E. Western blotting analysis showed that the protein levels of PSF, NONO, SNAI2, and GAPDH were detected in HCT116 empty vector, GAPLINC-siRNA, GAPLINC-pcDNA3.1, PSF-siRNA, and NONO-siRNA cells. Data represent mean ± standard deviation from three independent experiments. * P

Techniques Used: SDS Page, Mass Spectrometry, Western Blot, Negative Control, Reverse Transcription Polymerase Chain Reaction, Transwell Assay, shRNA, Transfection, Plasmid Preparation, Standard Deviation

6) Product Images from "LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3"

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0873-2

DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P
Figure Legend Snippet: DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P

Techniques Used: Expressing, Quantitative RT-PCR, Labeling, Incubation, SDS Page, Electrophoresis, Silver Staining, Western Blot, Isolation, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Over Expression

7) Product Images from "Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma"

Article Title: Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0472-6

MUC5B-AS1 increases the stability of MUC5B mRNA by forming a protective RNA duplex. a Schematic representation of the PCR amplification regions for overlapping (OL) and non-overlapping (non-OL) regions of MUC5B. We designed two pairs of primers to amplify the OL regions (OL1 and OL2) and non-OL (non-OL1 and non-OL2) regions of MUC5B, respectively. F forward primer, R reverse primer. b RT-PCR products of OL and non-OL regions of MUC5B. Total RNA samples were treated with RNAse A + T cocktail and then cleaned up RNA using RNeasy kits. RT-PCR was conducted using the primers to detect the OL and non-OL regions of the MUC5B mRNA. OL and non-OL regions of KRT7-AS were used as a positive control. c Stability of MUC5B mRNA over 12 h was measured by qRT-PCR relative to time 0 h after blocking new RNA synthesis with Actinomycin D (1 μg/mL; indicated with black arrow). H1299 cells with MUC5B-AS1 or empty vector stable expression were treated with 1 μg/mL ActD, and then harvested cells for RNA purification at 12 h after addition of ActD. Then, MUC5B mRNA stability were subsequently measured by qRT-PCR and were normalized against a synthesized exogenous reference λ polyA + RNA. Student’s t -test, * P
Figure Legend Snippet: MUC5B-AS1 increases the stability of MUC5B mRNA by forming a protective RNA duplex. a Schematic representation of the PCR amplification regions for overlapping (OL) and non-overlapping (non-OL) regions of MUC5B. We designed two pairs of primers to amplify the OL regions (OL1 and OL2) and non-OL (non-OL1 and non-OL2) regions of MUC5B, respectively. F forward primer, R reverse primer. b RT-PCR products of OL and non-OL regions of MUC5B. Total RNA samples were treated with RNAse A + T cocktail and then cleaned up RNA using RNeasy kits. RT-PCR was conducted using the primers to detect the OL and non-OL regions of the MUC5B mRNA. OL and non-OL regions of KRT7-AS were used as a positive control. c Stability of MUC5B mRNA over 12 h was measured by qRT-PCR relative to time 0 h after blocking new RNA synthesis with Actinomycin D (1 μg/mL; indicated with black arrow). H1299 cells with MUC5B-AS1 or empty vector stable expression were treated with 1 μg/mL ActD, and then harvested cells for RNA purification at 12 h after addition of ActD. Then, MUC5B mRNA stability were subsequently measured by qRT-PCR and were normalized against a synthesized exogenous reference λ polyA + RNA. Student’s t -test, * P

Techniques Used: Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Blocking Assay, Plasmid Preparation, Expressing, Purification, Synthesized

Identification of an overlapping antisense lncRNA at the MUC5B gene locus. a The localizations of MUC5B-AS1 and MUC5B on the UCSC genome browser. The schema was not drawn to scale. MUC5B-AS1 is located at chromosomal 11p15.5, and composes of two exons. MUC5B-AS1 is an antisense lncRNA, embedded on the opposite DNA strand of the MUC5B gene within its 31st exon. Green blocks indicate exons, and red blocks are overlapping regions. Primers of MUC5B-AS1 and MUC5B are also indicated in the schema: F primer forward primer, R primer reverse primer. The forward primer of MUC5B-AS1 spans the exon1–exon2 junction to avoid the non-specific amplification of MUC5B mRNA or genomic DNA. b Upper chart: ORF prediction of MUC5B-AS1 sequence. Three potential ORFs that might code peptides of 33–118 amino acids are present in the MUC5B-AS1 sequence. Lower chart: coding potentials of lncRNAs (MUC5B-AS1, MALAT1, TUG1) and mRNA (MUC5B, GAPDH, ACTB) were calculated using CPAT and CPC. c Localization of MUC5B-AS1 by RNA-FISH. Blue, DAPI-stained nuclei; red, Cy3-labeled positive hybridization signals (scale bar, 20 μm). The U6 and 18S were used as positive control. d Expression analysis of MUC5B-AS1 in lung adenocarcinoma tissues ( n = 72) and paired adjacent normal lung tissues ( n = 72). The ΔCt was used to show the expression level of MUC5B-AS1 (ΔCt = Ct MUC5B-AS1 –Ct β-actin ). Lower ΔCt values indicate higher expression. Normal vs. tumor tissues, Student’s t -test
Figure Legend Snippet: Identification of an overlapping antisense lncRNA at the MUC5B gene locus. a The localizations of MUC5B-AS1 and MUC5B on the UCSC genome browser. The schema was not drawn to scale. MUC5B-AS1 is located at chromosomal 11p15.5, and composes of two exons. MUC5B-AS1 is an antisense lncRNA, embedded on the opposite DNA strand of the MUC5B gene within its 31st exon. Green blocks indicate exons, and red blocks are overlapping regions. Primers of MUC5B-AS1 and MUC5B are also indicated in the schema: F primer forward primer, R primer reverse primer. The forward primer of MUC5B-AS1 spans the exon1–exon2 junction to avoid the non-specific amplification of MUC5B mRNA or genomic DNA. b Upper chart: ORF prediction of MUC5B-AS1 sequence. Three potential ORFs that might code peptides of 33–118 amino acids are present in the MUC5B-AS1 sequence. Lower chart: coding potentials of lncRNAs (MUC5B-AS1, MALAT1, TUG1) and mRNA (MUC5B, GAPDH, ACTB) were calculated using CPAT and CPC. c Localization of MUC5B-AS1 by RNA-FISH. Blue, DAPI-stained nuclei; red, Cy3-labeled positive hybridization signals (scale bar, 20 μm). The U6 and 18S were used as positive control. d Expression analysis of MUC5B-AS1 in lung adenocarcinoma tissues ( n = 72) and paired adjacent normal lung tissues ( n = 72). The ΔCt was used to show the expression level of MUC5B-AS1 (ΔCt = Ct MUC5B-AS1 –Ct β-actin ). Lower ΔCt values indicate higher expression. Normal vs. tumor tissues, Student’s t -test

Techniques Used: Amplification, Sequencing, Fluorescence In Situ Hybridization, Staining, Labeling, Hybridization, Positive Control, Expressing

8) Product Images from "Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica"

Article Title: Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica

Journal: Scientific Reports

doi: 10.1038/s41598-018-23997-w

Identification of aptamers against EhCFIm25. ( A ) Schematic representation of ssDNA oligonucleotides used to generate the ssRNA library for the SELEX protocol. ( B ) The predicted secondary structures of the C4 and C5 aptamers. ( C – G ) The RNA-electrophoretic mobility shift assay (REMSA). Proteins were incubated with a biotin-labelled RNA probe, and the RNA-protein complexes were resolved via PAGE and chemiluminescence assays. ( C ) A REMSA of the R7 aptamer population with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. Proteinase K (1 µg), unspecific competitor (tRNA) and RNA molecules from the first round of SELEX (R0) were used as controls. The image results from the grouping of gels cropped from different parts of the same gel and a different gel. ( D ) A REMSA of C4 and C5 aptamers with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. The image results from the grouping of gels cropped from two different gels. ( E ) A REMSA of C4 and C5 aptamers with E . histolytica protein extracts. Anti-EhCFIm25 antibodies were used as controls. ( F and G ) A REMSA of C4 and C5 aptamers with protein extracts from HeLa cells ( F ) and Trypanosoma cruzi parasites ( G ). Eh: E . histolytica ; Tc: T . cruzi ; single arrowhead: the RNA-protein complex; double arrowhead: the RNA-protein-antibody complex; asterisk: the free probe.
Figure Legend Snippet: Identification of aptamers against EhCFIm25. ( A ) Schematic representation of ssDNA oligonucleotides used to generate the ssRNA library for the SELEX protocol. ( B ) The predicted secondary structures of the C4 and C5 aptamers. ( C – G ) The RNA-electrophoretic mobility shift assay (REMSA). Proteins were incubated with a biotin-labelled RNA probe, and the RNA-protein complexes were resolved via PAGE and chemiluminescence assays. ( C ) A REMSA of the R7 aptamer population with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. Proteinase K (1 µg), unspecific competitor (tRNA) and RNA molecules from the first round of SELEX (R0) were used as controls. The image results from the grouping of gels cropped from different parts of the same gel and a different gel. ( D ) A REMSA of C4 and C5 aptamers with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. The image results from the grouping of gels cropped from two different gels. ( E ) A REMSA of C4 and C5 aptamers with E . histolytica protein extracts. Anti-EhCFIm25 antibodies were used as controls. ( F and G ) A REMSA of C4 and C5 aptamers with protein extracts from HeLa cells ( F ) and Trypanosoma cruzi parasites ( G ). Eh: E . histolytica ; Tc: T . cruzi ; single arrowhead: the RNA-protein complex; double arrowhead: the RNA-protein-antibody complex; asterisk: the free probe.

Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Polyacrylamide Gel Electrophoresis, Mutagenesis

9) Product Images from "LncRNA MIAT overexpression reduced neuron apoptosis in a neonatal rat model of hypoxic-ischemic injury through miR-211/GDNF"

Article Title: LncRNA MIAT overexpression reduced neuron apoptosis in a neonatal rat model of hypoxic-ischemic injury through miR-211/GDNF

Journal: Cell Cycle

doi: 10.1080/15384101.2018.1560202

The interaction between lncRNA MIAT and miR-211. (a) Bioinformatics software predicted the binding sites between MIAT and miR-211. (b) AGO2 antibody was used for RIP, and miR-211 or MIAT expression in AGO2 complex was detected by qRT-PCR. (c) RNA pull-down assay was used to detect AGO2 expression in MIAT pull-down complex. Loc, positive control of MIAT. miR-211 expression in MIAT pull-down complex was detected by qRT-PCR.
Figure Legend Snippet: The interaction between lncRNA MIAT and miR-211. (a) Bioinformatics software predicted the binding sites between MIAT and miR-211. (b) AGO2 antibody was used for RIP, and miR-211 or MIAT expression in AGO2 complex was detected by qRT-PCR. (c) RNA pull-down assay was used to detect AGO2 expression in MIAT pull-down complex. Loc, positive control of MIAT. miR-211 expression in MIAT pull-down complex was detected by qRT-PCR.

Techniques Used: Software, Binding Assay, Expressing, Quantitative RT-PCR, Pull Down Assay, Positive Control

10) Product Images from "Recurrence-Associated Long Non-coding RNA LNAPPCC Facilitates Colon Cancer Progression via Forming a Positive Feedback Loop with PCDH7"

Article Title: Recurrence-Associated Long Non-coding RNA LNAPPCC Facilitates Colon Cancer Progression via Forming a Positive Feedback Loop with PCDH7

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2020.03.017

LNAPPCC Activates PCDH7 Expression via Binding EZH2 (A) RNA pull-down assay using biotin-labeled LNAPPCC was undertaken to detect the interaction between LNAPPCC and EZH2. (B) RIP assay using EZH2 specific antibody was undertaken to detect the binding between EZH2 and LNAPPCC. (C) Schematic outlining the predicted G-rich motif in LNAPPCC and the wild-type (WT) and mutated LNAPPCC sequences used for RIP with anti-EZH2. LNAPPCC-Mut contains a deletion of the G-rich motif. (D) After transient transfection of LNAPPCC-WT or LNAPPCC-Mut overexpression plasmids into HCT116 cells, RIP assay was undertaken using EZH2 specific antibody. (E) ChIP assay using EZH2 and H3K27me3 specific antibodies was undertaken in LNAPPCC stably overexpressed and control SW620 cells to detect the effects of LNAPPCC overexpression on the binding of EZH2 to PCDH7 promoter and H3K27me3 level at PCDH7 promoter. (F) ChIP assay using EZH2 and H3K27me3 specific antibodies was undertaken in LNAPPCC stably silenced and control HCT116 cells to detect the effects of LNAPPCC silencing on the binding of EZH2 to PCDH7 promoter and H3K27me3 level at PCDH7 promoter. (G) PCDH7 mRNA and protein levels in LNAPPCC stably overexpressed and control HCT116 cells were detected by quantitative real-time PCR and western blot. (H) PCDH7 mRNA and protein levels in LNAPPCC stably overexpressed and control SW620 cells were detected by quantitative real-time PCR and western blot. (I) PCDH7 mRNA and protein levels in LNAPPCC stably silenced and control HCT116 cells were detected by quantitative real-time PCR and western blot. Data are presented as mean ± SD based on at least three independent biological repeats. ∗p
Figure Legend Snippet: LNAPPCC Activates PCDH7 Expression via Binding EZH2 (A) RNA pull-down assay using biotin-labeled LNAPPCC was undertaken to detect the interaction between LNAPPCC and EZH2. (B) RIP assay using EZH2 specific antibody was undertaken to detect the binding between EZH2 and LNAPPCC. (C) Schematic outlining the predicted G-rich motif in LNAPPCC and the wild-type (WT) and mutated LNAPPCC sequences used for RIP with anti-EZH2. LNAPPCC-Mut contains a deletion of the G-rich motif. (D) After transient transfection of LNAPPCC-WT or LNAPPCC-Mut overexpression plasmids into HCT116 cells, RIP assay was undertaken using EZH2 specific antibody. (E) ChIP assay using EZH2 and H3K27me3 specific antibodies was undertaken in LNAPPCC stably overexpressed and control SW620 cells to detect the effects of LNAPPCC overexpression on the binding of EZH2 to PCDH7 promoter and H3K27me3 level at PCDH7 promoter. (F) ChIP assay using EZH2 and H3K27me3 specific antibodies was undertaken in LNAPPCC stably silenced and control HCT116 cells to detect the effects of LNAPPCC silencing on the binding of EZH2 to PCDH7 promoter and H3K27me3 level at PCDH7 promoter. (G) PCDH7 mRNA and protein levels in LNAPPCC stably overexpressed and control HCT116 cells were detected by quantitative real-time PCR and western blot. (H) PCDH7 mRNA and protein levels in LNAPPCC stably overexpressed and control SW620 cells were detected by quantitative real-time PCR and western blot. (I) PCDH7 mRNA and protein levels in LNAPPCC stably silenced and control HCT116 cells were detected by quantitative real-time PCR and western blot. Data are presented as mean ± SD based on at least three independent biological repeats. ∗p

Techniques Used: Expressing, Binding Assay, Pull Down Assay, Labeling, Transfection, Over Expression, Chromatin Immunoprecipitation, Stable Transfection, Real-time Polymerase Chain Reaction, Western Blot

11) Product Images from "LncSox4 promotes the self-renewal of liver tumour-initiating cells through Stat3-mediated Sox4 expression"

Article Title: LncSox4 promotes the self-renewal of liver tumour-initiating cells through Stat3-mediated Sox4 expression

Journal: Nature Communications

doi: 10.1038/ncomms12598

LncSox4 drives liver TIC self-renewal through Sox4. ( a , b ) LncSox4 is required for Sox4 expression. mRNA expression of the indicated genes in LncSox4 -silenced (sh LncSox4 ) and control cells (shCtrl) were examined using real-time PCR ( a ). Impaired Sox4 expression in LncSox4 -silenced cells was confirmed by western blot ( b ). ( c , d ) LncSox4 drives liver TIC self-renewal through Sox4 expression. Sox4 expression was rescued using PBPLV lentivirus in LncSox4 -silenced cells, and then sphere formation ( c ) and tumour initiation ( d ) assays were performed. Five thousand indicated primary HCC cells were used for sphere formation, and 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 cells were subcutaneously injected into BALB/c nude mice for tumour initiation. ( e ) Primary HCC cells were treated with CRISPR/Cas9 lentivirus for Sox4 deficiency, followed by LncSox4 overexpression (oeLnc). The indicated cells were used for in vivo tumour initiation, and the ratios of tumour-free mice were calculated 3 months later. ( f ) LncSox4 binds to Sox4 promoter. RNA ChIP assay was performed and fold enrichment was examined using real-time PCR. ( g , h ) The interaction of LncSox4 and Sox4 promoter was required for LncSox4 function. The Sox4 promoter region for LncSox4 binding (−3,647∼−3,537) was deleted using CRISPR/Cas9 approach ( Sox4PKO ), followed by LncSox4 overexpression. The established cells were examined for Sox4 expression and sphere formation capacities. Six samples were examined and similar results were found. Scale bars, C, H 500 μm. Data were shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis. * P
Figure Legend Snippet: LncSox4 drives liver TIC self-renewal through Sox4. ( a , b ) LncSox4 is required for Sox4 expression. mRNA expression of the indicated genes in LncSox4 -silenced (sh LncSox4 ) and control cells (shCtrl) were examined using real-time PCR ( a ). Impaired Sox4 expression in LncSox4 -silenced cells was confirmed by western blot ( b ). ( c , d ) LncSox4 drives liver TIC self-renewal through Sox4 expression. Sox4 expression was rescued using PBPLV lentivirus in LncSox4 -silenced cells, and then sphere formation ( c ) and tumour initiation ( d ) assays were performed. Five thousand indicated primary HCC cells were used for sphere formation, and 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 cells were subcutaneously injected into BALB/c nude mice for tumour initiation. ( e ) Primary HCC cells were treated with CRISPR/Cas9 lentivirus for Sox4 deficiency, followed by LncSox4 overexpression (oeLnc). The indicated cells were used for in vivo tumour initiation, and the ratios of tumour-free mice were calculated 3 months later. ( f ) LncSox4 binds to Sox4 promoter. RNA ChIP assay was performed and fold enrichment was examined using real-time PCR. ( g , h ) The interaction of LncSox4 and Sox4 promoter was required for LncSox4 function. The Sox4 promoter region for LncSox4 binding (−3,647∼−3,537) was deleted using CRISPR/Cas9 approach ( Sox4PKO ), followed by LncSox4 overexpression. The established cells were examined for Sox4 expression and sphere formation capacities. Six samples were examined and similar results were found. Scale bars, C, H 500 μm. Data were shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Mouse Assay, CRISPR, Over Expression, In Vivo, Chromatin Immunoprecipitation, Binding Assay, Two Tailed Test

LncSox4 interacts with Stat3. ( a ) RNA pull-down assay was performed using LncSox4 , Lnc-AS ( LncSox4 antisense RNA) and control RNA, and the samples were separated by Coomassie staining, followed by identification using mass spectrum. The black arrow indicates Stat3. KD, kiloDalton. ( b ) The interaction between LncSox4 and Stat3 was confirmed by RNA pull-down and western blot. Lnc-AS, LncSox4 anti-sense. β-Actin served as loading controls. ( c ) RIP was performed using anti-Stat3 and control IgG antibodies, followed by real-time PCR to examine the enrichment of LncSox4 and GAPDH. GAPDH served as a negative control. ( d ) LncSox4 is co-localized with Stat3. LncSox4 probes and anti-Stat3 antibody were used to examine the subcellular location using flow cytometer sorted CD133 - (Non-TIC), CD133 + (TIC) cells and stem-like oncospheres (Sphere). Scale bars, 10 μm. ( e ) Three regions of LncSox4 were examined for interaction with Stat3. The indicated truncates of LncSox4 were constructed and RNA pull-down assays were performed. The samples were examined by western blot with anti-Stat3 antibody. ( f ) LncSox4 interacts with Stat3. Biotin-labelled LncSox4 was obtained by in vitro transcription assays and incubated with recombination Stat3 protein, followed by RNA EMSA. 100 × unlabelled probes were used for competitive EMSA. ( g ) Bead-based proximity assay of LncSox4 , LncSox4 truncate (-#3 region) and LncSox4 -antisense (Lnc-AS ) (50 nnol) were incubated with various concentrations (horizontal axis) of Stat3 (left) or Stat3 (50 ng μl −1 ) mixed with various concentrations (horizontal axis) of biotin-labelled RNA (right); and units generated by the assay system were shown. ( h ) LncSox4 (FL), LncSox4 truncate (-#3 region) overexpressing primary HCC cells were established, followed by sphere-formation assay. Typical spheres were shown in left panels, and sphere formation ratios in right panels. Scale bars, 500 μm. Data were shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis. * P
Figure Legend Snippet: LncSox4 interacts with Stat3. ( a ) RNA pull-down assay was performed using LncSox4 , Lnc-AS ( LncSox4 antisense RNA) and control RNA, and the samples were separated by Coomassie staining, followed by identification using mass spectrum. The black arrow indicates Stat3. KD, kiloDalton. ( b ) The interaction between LncSox4 and Stat3 was confirmed by RNA pull-down and western blot. Lnc-AS, LncSox4 anti-sense. β-Actin served as loading controls. ( c ) RIP was performed using anti-Stat3 and control IgG antibodies, followed by real-time PCR to examine the enrichment of LncSox4 and GAPDH. GAPDH served as a negative control. ( d ) LncSox4 is co-localized with Stat3. LncSox4 probes and anti-Stat3 antibody were used to examine the subcellular location using flow cytometer sorted CD133 - (Non-TIC), CD133 + (TIC) cells and stem-like oncospheres (Sphere). Scale bars, 10 μm. ( e ) Three regions of LncSox4 were examined for interaction with Stat3. The indicated truncates of LncSox4 were constructed and RNA pull-down assays were performed. The samples were examined by western blot with anti-Stat3 antibody. ( f ) LncSox4 interacts with Stat3. Biotin-labelled LncSox4 was obtained by in vitro transcription assays and incubated with recombination Stat3 protein, followed by RNA EMSA. 100 × unlabelled probes were used for competitive EMSA. ( g ) Bead-based proximity assay of LncSox4 , LncSox4 truncate (-#3 region) and LncSox4 -antisense (Lnc-AS ) (50 nnol) were incubated with various concentrations (horizontal axis) of Stat3 (left) or Stat3 (50 ng μl −1 ) mixed with various concentrations (horizontal axis) of biotin-labelled RNA (right); and units generated by the assay system were shown. ( h ) LncSox4 (FL), LncSox4 truncate (-#3 region) overexpressing primary HCC cells were established, followed by sphere-formation assay. Typical spheres were shown in left panels, and sphere formation ratios in right panels. Scale bars, 500 μm. Data were shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis. * P

Techniques Used: Pull Down Assay, Staining, Western Blot, Real-time Polymerase Chain Reaction, Negative Control, Flow Cytometry, Cytometry, Construct, In Vitro, Incubation, Proximity Assay, Generated, Tube Formation Assay, Two Tailed Test

12) Product Images from "linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis"

Article Title: linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis

Journal: Genes & Development

doi: 10.1101/gad.217018.113

Identification and characterization of linc-HOXA1 as a noncoding RNA. ( A ) Illustration of the genomic location and transcript structure of the linc-HOXA1 gene and its three isoforms. ( B ) Cumulative probability distribution of coding potential as measured
Figure Legend Snippet: Identification and characterization of linc-HOXA1 as a noncoding RNA. ( A ) Illustration of the genomic location and transcript structure of the linc-HOXA1 gene and its three isoforms. ( B ) Cumulative probability distribution of coding potential as measured

Techniques Used:

Knockdown of linc-HOXA1 increases Hoxa1 levels. ( A ) Bar graphs showing changes in average number of linc-HOXA1 and Hoxa1 RNA molecules per cell upon treatment with siRNA or Isis antisense oligonucleotides targeting either linc-HOXA1 ( top row) or Hoxa1
Figure Legend Snippet: Knockdown of linc-HOXA1 increases Hoxa1 levels. ( A ) Bar graphs showing changes in average number of linc-HOXA1 and Hoxa1 RNA molecules per cell upon treatment with siRNA or Isis antisense oligonucleotides targeting either linc-HOXA1 ( top row) or Hoxa1

Techniques Used:

Overexpression of linc-HOXA1 isoforms does not alter Hoxa1 abundance. ( A ) Bar plots showing the number of the various linc-HOXA1 RNA isoforms overexpressed in embryonic stem cells (and a vector containing GFP as a control) ( left ) and the resultant number
Figure Legend Snippet: Overexpression of linc-HOXA1 isoforms does not alter Hoxa1 abundance. ( A ) Bar plots showing the number of the various linc-HOXA1 RNA isoforms overexpressed in embryonic stem cells (and a vector containing GFP as a control) ( left ) and the resultant number

Techniques Used: Over Expression, Plasmid Preparation

Detection of individual RNA isoforms of linc-HOXA1 in single cells. ( A ) Raw micrographs of a single cell containing all three isoforms. (Three left panels) We designed probes specifically targeting exons 3c, 3b, and 3a, each of which revealed single spots
Figure Legend Snippet: Detection of individual RNA isoforms of linc-HOXA1 in single cells. ( A ) Raw micrographs of a single cell containing all three isoforms. (Three left panels) We designed probes specifically targeting exons 3c, 3b, and 3a, each of which revealed single spots

Techniques Used:

13) Product Images from "Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR"

Article Title: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki797

Depletion of KSRP enhances iNOS 3′-UTR RNA stability in in vitro degradation assays. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with the CM for 6 h and cytoplasmic proteins were isolated. Parts of these extracts were immunodepleted of KSRP by using a polyclonal anti-KSRP antibody (KSRP-depl). The extracts were incubated with radiolabeled 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNA with (iNOS 3′-UTR) or without (non-AU) the AREs for different time periods from 0 to 30 min. Then the degradation of the RNA in the different samples was stopped by adding SDS. To normalize the subsequent purification steps in vitro transcribed radiolabeled β-actin RNA fragments were added. After phenol extraction and ethanol precipitation the material was separated on denaturing urea polyacrylamide gels. ( A ) Scheme of the human 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs used for in vitro decay experiments. The positions of AU-repeats are indicated by arrowheads. ( B ) The degree of KSRP depletion of two different immunodepletion reactions (IP α-KSRP) were analyzed by western blotting using specific anti-KSRP- and anti-β-tubulin antibodies. For comparison the corresponding untreated samples were analyzed. The position of KSRP and β-tubulin is indicated. ( C ) Summary of densitometric analyses of eight different in vitro decay assays using 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs containing the AREs (iNOS 3′-UTR). Data points (means ± SEM) represent relative iNOS 3′-UTR RNA levels (100% = 0 min incubation; circles: undepleted extracts from CM-treated cells; squares: KSRP-depleted extracts from CM-treated cells; ** P
Figure Legend Snippet: Depletion of KSRP enhances iNOS 3′-UTR RNA stability in in vitro degradation assays. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with the CM for 6 h and cytoplasmic proteins were isolated. Parts of these extracts were immunodepleted of KSRP by using a polyclonal anti-KSRP antibody (KSRP-depl). The extracts were incubated with radiolabeled 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNA with (iNOS 3′-UTR) or without (non-AU) the AREs for different time periods from 0 to 30 min. Then the degradation of the RNA in the different samples was stopped by adding SDS. To normalize the subsequent purification steps in vitro transcribed radiolabeled β-actin RNA fragments were added. After phenol extraction and ethanol precipitation the material was separated on denaturing urea polyacrylamide gels. ( A ) Scheme of the human 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs used for in vitro decay experiments. The positions of AU-repeats are indicated by arrowheads. ( B ) The degree of KSRP depletion of two different immunodepletion reactions (IP α-KSRP) were analyzed by western blotting using specific anti-KSRP- and anti-β-tubulin antibodies. For comparison the corresponding untreated samples were analyzed. The position of KSRP and β-tubulin is indicated. ( C ) Summary of densitometric analyses of eight different in vitro decay assays using 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs containing the AREs (iNOS 3′-UTR). Data points (means ± SEM) represent relative iNOS 3′-UTR RNA levels (100% = 0 min incubation; circles: undepleted extracts from CM-treated cells; squares: KSRP-depleted extracts from CM-treated cells; ** P

Techniques Used: In Vitro, Incubation, Isolation, Purification, Ethanol Precipitation, Western Blot

Cytokine incubation reduces intracellular binding of KSRP and enhances intracellular binding of HuR to the iNOS mRNA. DLD-1 cells were incubated for 4 h with or without CM. Cells were lyzed and RNA bound by KSRP or HuR protein was immunoprecipitated by specific antibodies. Immunoprecipitation with IgG was used as negative control. To normalize for the subsequent RNA purification steps 1 ng/sample of luciferase RNA transcribed in vitro was added before the RNA was isolated from immunoprecipitated proteins. The amount of iNOS mRNA bound by KSRP or HuR was determined by qRT–PCR using the luciferase RNA as normalization control. The values of the IgG controls were subtracted and the values for the iNOS mRNA bound by HuR were divided by the values for the iNOS mRNA bound by KSRP. A summary of 12 immunoprecipitation-qRT–PCR analyses is shown. Columns (means ± SEM) represent relative iNOS mRNA levels bound by HuR divided by the relative iNOS mRNA levels bound by KSRP ( ** P
Figure Legend Snippet: Cytokine incubation reduces intracellular binding of KSRP and enhances intracellular binding of HuR to the iNOS mRNA. DLD-1 cells were incubated for 4 h with or without CM. Cells were lyzed and RNA bound by KSRP or HuR protein was immunoprecipitated by specific antibodies. Immunoprecipitation with IgG was used as negative control. To normalize for the subsequent RNA purification steps 1 ng/sample of luciferase RNA transcribed in vitro was added before the RNA was isolated from immunoprecipitated proteins. The amount of iNOS mRNA bound by KSRP or HuR was determined by qRT–PCR using the luciferase RNA as normalization control. The values of the IgG controls were subtracted and the values for the iNOS mRNA bound by HuR were divided by the values for the iNOS mRNA bound by KSRP. A summary of 12 immunoprecipitation-qRT–PCR analyses is shown. Columns (means ± SEM) represent relative iNOS mRNA levels bound by HuR divided by the relative iNOS mRNA levels bound by KSRP ( ** P

Techniques Used: Incubation, Binding Assay, Immunoprecipitation, Negative Control, Purification, Luciferase, In Vitro, Isolation, Quantitative RT-PCR

Binding of KSRP and HuR to the 3′-UTR of the human iNOS mRNA is mutually exclusive. 32 P-radiolabeled in vitro transcribed RNAs containing the sequence of subfragment C (387–477) were incubated in parallel with fixed amounts (0.6 µg) of GST-HuR or GST-KSRP and increasing amounts of GST-KSRP or GST-HuR (0 to 1.2 µg), respectively. After binding, proteins were UV crosslinked to the RNA and the complexes were digested with RNase. RNA–protein complexes were separated on SDS–polyacrylamide gels. The positions of RNA–protein complexes are indicated. (A) Radiolabeled fragment C-RNAs were incubated with 0.6 µg GST-HuR (0.6 µg) and 0 to 1.2 µg GST-KSRP. (B) Radiolabeled fragment C-RNAs were incubated with 0.6 µg GST-KSRP (0.6 µg) and 0–1.2 µg GST-HuR.
Figure Legend Snippet: Binding of KSRP and HuR to the 3′-UTR of the human iNOS mRNA is mutually exclusive. 32 P-radiolabeled in vitro transcribed RNAs containing the sequence of subfragment C (387–477) were incubated in parallel with fixed amounts (0.6 µg) of GST-HuR or GST-KSRP and increasing amounts of GST-KSRP or GST-HuR (0 to 1.2 µg), respectively. After binding, proteins were UV crosslinked to the RNA and the complexes were digested with RNase. RNA–protein complexes were separated on SDS–polyacrylamide gels. The positions of RNA–protein complexes are indicated. (A) Radiolabeled fragment C-RNAs were incubated with 0.6 µg GST-HuR (0.6 µg) and 0 to 1.2 µg GST-KSRP. (B) Radiolabeled fragment C-RNAs were incubated with 0.6 µg GST-KSRP (0.6 µg) and 0–1.2 µg GST-HuR.

Techniques Used: Binding Assay, In Vitro, Sequencing, Incubation

Overexpression of a KSRP mutant which is not able to bind to RNA enhances iNOS expression. Plasmids allowing high level expression of a mutant KSRP protein not able to bind to RNA (KSRPdel) were stably transfected into DLD-1 cells. Cells transfected with the pcDNA3 vector backbone (pcDNA3) were used as controls. For analysis of iNOS expression pools of stable transfected cell were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with (CM) or without (Co) the cytokine mixture for 6 h, RNA was isolated and iNOS and GAPDH mRNA expression was analyzed by real-time RT–PCR. To determine iNOS-mediated NO-production cells were incubated for 24 h with or without CM and the supernatant of the cells was analyzed for nitrite content. ( A ) Pooled populations of pcDNA3- or pcDNA-KSRPdel cells were analyzed for KSRP expression by western blots using specific anti-KSRP antibodies. One representative blot out of three is shown. ( B ) A summary of 5 qRT–PCR analyses is shown using RNAs from DLD-1-pcDNA3 (pcDNA3) or DLD-1-pcDNA3-KSRPdel (KSRPdel) cells. Data (means ± SEM) represent relative iNOS mRNA levels ( * P
Figure Legend Snippet: Overexpression of a KSRP mutant which is not able to bind to RNA enhances iNOS expression. Plasmids allowing high level expression of a mutant KSRP protein not able to bind to RNA (KSRPdel) were stably transfected into DLD-1 cells. Cells transfected with the pcDNA3 vector backbone (pcDNA3) were used as controls. For analysis of iNOS expression pools of stable transfected cell were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with (CM) or without (Co) the cytokine mixture for 6 h, RNA was isolated and iNOS and GAPDH mRNA expression was analyzed by real-time RT–PCR. To determine iNOS-mediated NO-production cells were incubated for 24 h with or without CM and the supernatant of the cells was analyzed for nitrite content. ( A ) Pooled populations of pcDNA3- or pcDNA-KSRPdel cells were analyzed for KSRP expression by western blots using specific anti-KSRP antibodies. One representative blot out of three is shown. ( B ) A summary of 5 qRT–PCR analyses is shown using RNAs from DLD-1-pcDNA3 (pcDNA3) or DLD-1-pcDNA3-KSRPdel (KSRPdel) cells. Data (means ± SEM) represent relative iNOS mRNA levels ( * P

Techniques Used: Over Expression, Mutagenesis, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Isolation, Quantitative RT-PCR, Western Blot

Analysis of the KSRP binding site in the human iNOS 3′-UTR RNA. Purified BSA, glutathione-S-transferase (GST), and GST-KSRP or GST-HuR fusion proteins were incubated with different radiolabeled RNAs generated by in vitro transcription using the different iNOS 3′-UTR fragments shown in Figure A. After binding, proteins were UV crosslinked to the RNA and the complexes were digested with RNase. RNA–protein complexes were separated on SDS–polyacrylamide gels. ( A ) Structure of the human iNOS 3′-UTR mRNA and fragments used in RNA binding studies. Scheme of the human iNOS 3′-UTR mRNA (477 nt) and transcripts used in RNA binding studies. The initial UGA nucleotide sequence (−3 to −1) corresponds to the translation termination codon. AUUUA and AUUUUA repeats are indicated by arrowheads. The sequences of different mutations in fragment C are shown. ( B ) KSRP binds to the AU-fragment of the human iNOS 3′-UTR. 32 P-radiolabeled RNA transcripts [3′-UTR; non-AU; AU; see (A)] were incubated with BSA, GST, GST-KSRP fusion protein (KSRP) or GST-HuR fusion protein (HuR). The positions of RNA–protein complexes are indicated. ( C ) KSRP binds to the most 3′-located ARE in the human iNOS 3′-UTR. 32 P-radiolabeled 3′-UTR, subfragment A (232–319, frag A), subfragment B (317–420; frag B) or subfragment C (387–477; frag C) were incubated with GST-KSRP (GST-KSRP) protein. The positions of RNA–protein complexes are indicated (left side). 32 P-radiolabeled RNA transcripts (5′-ARE mutated: mut 1; 3′-ARE mutated: mut 2; both AREs mutated: mut 1 + 2) were incubated with either GST or GST-KSRP fusion protein (KSRP). The positions of RNA–protein complexes are indicated (right side).
Figure Legend Snippet: Analysis of the KSRP binding site in the human iNOS 3′-UTR RNA. Purified BSA, glutathione-S-transferase (GST), and GST-KSRP or GST-HuR fusion proteins were incubated with different radiolabeled RNAs generated by in vitro transcription using the different iNOS 3′-UTR fragments shown in Figure A. After binding, proteins were UV crosslinked to the RNA and the complexes were digested with RNase. RNA–protein complexes were separated on SDS–polyacrylamide gels. ( A ) Structure of the human iNOS 3′-UTR mRNA and fragments used in RNA binding studies. Scheme of the human iNOS 3′-UTR mRNA (477 nt) and transcripts used in RNA binding studies. The initial UGA nucleotide sequence (−3 to −1) corresponds to the translation termination codon. AUUUA and AUUUUA repeats are indicated by arrowheads. The sequences of different mutations in fragment C are shown. ( B ) KSRP binds to the AU-fragment of the human iNOS 3′-UTR. 32 P-radiolabeled RNA transcripts [3′-UTR; non-AU; AU; see (A)] were incubated with BSA, GST, GST-KSRP fusion protein (KSRP) or GST-HuR fusion protein (HuR). The positions of RNA–protein complexes are indicated. ( C ) KSRP binds to the most 3′-located ARE in the human iNOS 3′-UTR. 32 P-radiolabeled 3′-UTR, subfragment A (232–319, frag A), subfragment B (317–420; frag B) or subfragment C (387–477; frag C) were incubated with GST-KSRP (GST-KSRP) protein. The positions of RNA–protein complexes are indicated (left side). 32 P-radiolabeled RNA transcripts (5′-ARE mutated: mut 1; 3′-ARE mutated: mut 2; both AREs mutated: mut 1 + 2) were incubated with either GST or GST-KSRP fusion protein (KSRP). The positions of RNA–protein complexes are indicated (right side).

Techniques Used: Binding Assay, Purification, Incubation, Generated, In Vitro, RNA Binding Assay, Sequencing

Modulation of KSRP expression alters cytokine-induced iNOS mRNA expression and iNOS-dependent NO-production. Plasmid constructs allowing high level expression of sense (pcDNA-His-KSRP) or antisense (pcDNA-KSRPas) KSRP cDNA were stably transfected into DLD-1 cells. Cells transfected with the pcDNA3 vector backbone (pcDNA3) were used as controls. For analysis of iNOS expression pools of stable transfected cell were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with (CM) or without (Co) the cytokine mixture for 6 h, RNA was isolated and iNOS and GAPDH mRNA expression was analyzed. To determine iNOS-mediated NO-production cells were incubated for 24 h with or without CM and the supernatant of the cells was analyzed for nitrite content. As another approach to downregulate KSRP expression the RNA interference technique was used. DLD-1 cells were transfected with siRNA directed against luciferase (control, siLUC) or KSRP (siKSRP). After 24 h the transfected cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with or without CM for 6 h, RNA was isolated and iNOS, KSRP and GAPDH mRNA expression was analyzed by real-time RT–PCR. ( A ) Western blots using specific anti-KSRP- and anti-β-tubulin antibodies and extracts from the stable transfected DLD-1 cell pools. The blots are representative of four other blots showing similar results. The positions of KSRP and β-tubulin are indicated. ( B ) A summary of 10 qRT–PCR analyses is shown using RNAs form DLD-1-pcDNA3 (pcDNA3), DLD-1-pcDNA-His-KSRP (pcDNA-His-KSRP) or DLD-1-pcDNA3-KSRPas (pcDNA-KSRPas) cells. Data (means ± SEM) represent relative iNOS mRNA levels ( *** P
Figure Legend Snippet: Modulation of KSRP expression alters cytokine-induced iNOS mRNA expression and iNOS-dependent NO-production. Plasmid constructs allowing high level expression of sense (pcDNA-His-KSRP) or antisense (pcDNA-KSRPas) KSRP cDNA were stably transfected into DLD-1 cells. Cells transfected with the pcDNA3 vector backbone (pcDNA3) were used as controls. For analysis of iNOS expression pools of stable transfected cell were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with (CM) or without (Co) the cytokine mixture for 6 h, RNA was isolated and iNOS and GAPDH mRNA expression was analyzed. To determine iNOS-mediated NO-production cells were incubated for 24 h with or without CM and the supernatant of the cells was analyzed for nitrite content. As another approach to downregulate KSRP expression the RNA interference technique was used. DLD-1 cells were transfected with siRNA directed against luciferase (control, siLUC) or KSRP (siKSRP). After 24 h the transfected cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with or without CM for 6 h, RNA was isolated and iNOS, KSRP and GAPDH mRNA expression was analyzed by real-time RT–PCR. ( A ) Western blots using specific anti-KSRP- and anti-β-tubulin antibodies and extracts from the stable transfected DLD-1 cell pools. The blots are representative of four other blots showing similar results. The positions of KSRP and β-tubulin are indicated. ( B ) A summary of 10 qRT–PCR analyses is shown using RNAs form DLD-1-pcDNA3 (pcDNA3), DLD-1-pcDNA-His-KSRP (pcDNA-His-KSRP) or DLD-1-pcDNA3-KSRPas (pcDNA-KSRPas) cells. Data (means ± SEM) represent relative iNOS mRNA levels ( *** P

Techniques Used: Expressing, Plasmid Preparation, Construct, Stable Transfection, Transfection, Incubation, Isolation, Luciferase, Quantitative RT-PCR, Western Blot

Purification of RNA-bps interacting with the 3′-UTR of the human iNOS mRNA. To purify proteins binding to the 3′-UTR of the human iNOS mRNA affinity chromatographies using biotinylated iNOS 3′-UTR RNA were performed ( 45 , 46 ) as described in Materials and Methods. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with the cytokine mixture for 6 h and protein extracts were isolated. These extracts were incubated with biotinylated iNOS 3′-UTR RNA (3′-UTR) or biotinylated iNOS 3′-UTR RNA without the ARE-sequences (3′-UTR Δ–ARE) and streptavidine-agarose beads. After several washing and centrifugation steps the RNA-bps were eluted by 2 M KCl. ( A ) Proofing the applicability of this method western blots using a specific anti-HuR antibody were performed, since HuR is known to interact with the human iNOS 3′-UTR ( 28 ). As a positive control bacterial expressed GST-HuR fusion protein was also loaded on the SDS gel. One representative blot of three different experiments were shown. ( B ) The eluates were tested for the presence of KSRP by western blots using a specific anti-KSRP antibody. As a positive control bacterial expressed His-KSRP fusion protein was also loaded on the SDS gel. One representative blot of three different experiments were shown.
Figure Legend Snippet: Purification of RNA-bps interacting with the 3′-UTR of the human iNOS mRNA. To purify proteins binding to the 3′-UTR of the human iNOS mRNA affinity chromatographies using biotinylated iNOS 3′-UTR RNA were performed ( 45 , 46 ) as described in Materials and Methods. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then cells were incubated with the cytokine mixture for 6 h and protein extracts were isolated. These extracts were incubated with biotinylated iNOS 3′-UTR RNA (3′-UTR) or biotinylated iNOS 3′-UTR RNA without the ARE-sequences (3′-UTR Δ–ARE) and streptavidine-agarose beads. After several washing and centrifugation steps the RNA-bps were eluted by 2 M KCl. ( A ) Proofing the applicability of this method western blots using a specific anti-HuR antibody were performed, since HuR is known to interact with the human iNOS 3′-UTR ( 28 ). As a positive control bacterial expressed GST-HuR fusion protein was also loaded on the SDS gel. One representative blot of three different experiments were shown. ( B ) The eluates were tested for the presence of KSRP by western blots using a specific anti-KSRP antibody. As a positive control bacterial expressed His-KSRP fusion protein was also loaded on the SDS gel. One representative blot of three different experiments were shown.

Techniques Used: Purification, Binding Assay, Incubation, Isolation, Centrifugation, Western Blot, Positive Control, SDS-Gel

14) Product Images from "Characterization of antennal chemosensilla and associated odorant binding as well as chemosensory proteins in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae)"

Article Title: Characterization of antennal chemosensilla and associated odorant binding as well as chemosensory proteins in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae)

Journal: Scientific Reports

doi: 10.1038/s41598-018-25996-3

In situ hybridization of OBP3 and OBP18 in antennae of M. mediator . Dig-labelled antisense RNA probes for OBPs were hybridized to longitudinal sections through the female and male antennae and visualized by red fluorescence. OBP3 probe labeled cells were located at the base of the s. basiconica type 1 and type 3 in female antennae. In male antennae, the labeled cells were located at the base of s. basiconica type 1. OBP18 positive cells were located at the base of the s. basiconica type 1 on female and male antennae.
Figure Legend Snippet: In situ hybridization of OBP3 and OBP18 in antennae of M. mediator . Dig-labelled antisense RNA probes for OBPs were hybridized to longitudinal sections through the female and male antennae and visualized by red fluorescence. OBP3 probe labeled cells were located at the base of the s. basiconica type 1 and type 3 in female antennae. In male antennae, the labeled cells were located at the base of s. basiconica type 1. OBP18 positive cells were located at the base of the s. basiconica type 1 on female and male antennae.

Techniques Used: In Situ Hybridization, Fluorescence, Labeling

15) Product Images from "Long noncoding RNA GAS5-AS1 suppresses growth and metastasis of cervical cancer by increasing GAS5 stability"

Article Title: Long noncoding RNA GAS5-AS1 suppresses growth and metastasis of cervical cancer by increasing GAS5 stability

Journal: American Journal of Translational Research

doi:

GAS5-AS1 decreases the m 6 A modification of GAS5. A. MeRIP-qPCR analysis of fragmented GAS5 RNA in Caski with GAS5-AS1 or mutant GAS5-AS1 overexpression. B. MeRIP-qPCR analysis of fragmented GAS5 RNA in HeLa with GAS5-AS1 knockdown. C. RIP assay was performed
Figure Legend Snippet: GAS5-AS1 decreases the m 6 A modification of GAS5. A. MeRIP-qPCR analysis of fragmented GAS5 RNA in Caski with GAS5-AS1 or mutant GAS5-AS1 overexpression. B. MeRIP-qPCR analysis of fragmented GAS5 RNA in HeLa with GAS5-AS1 knockdown. C. RIP assay was performed

Techniques Used: Modification, Real-time Polymerase Chain Reaction, Mutagenesis, Over Expression

16) Product Images from "Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) Promotes Hepatocellular Carcinoma Progression Through Interaction with High-Mobility Group AT-hook 2 and MiR-4516"

Article Title: Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) Promotes Hepatocellular Carcinoma Progression Through Interaction with High-Mobility Group AT-hook 2 and MiR-4516

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.911179

LSINCT5 acts as a ceRNA of miR-4516 to promote HCC progression. ( A ) RIP assays followed by qPCR to identify putative miRNAs associated with LSINCT5. ( B ) The miRNAs were verified by RNA pulldown a with biotin-labeled sense or antisense LSINCT5. ( C ) Luciferase reporter assays identified miR-4516 could interact with LSINCT5 (top). RNA pulldown assay with anti-AGO2 was performed in HepG2 cells with miR-4516 overexpression followed by qPCR to enrich LSINCT5 (bottom). ( D ) The predicted base-pairing between LSINCT5 and miR-4516 at miRDB. Three hits were detected. ( E ) RNA-FISH assay revealed the co-localization of LSINCT5 (Alexa Fluor 488, green dots) and miR-4516 (Cy3, red dots) in HepG2 cells. Scale bar, 5 μm. ( F ) CCK-8 viability assay for Huh7 cells and HepG2 cells transfected with plasmids as indicated. ( G ) Tumor sphere formation assay for HepG2 cells transfected with negative control (NC), miR-4516 mimics + oe Vec or miR-4516 mimics + LSINCT5. Quantification results were shown on the right panel. ( H ) Western blot assays for BclxL, STAT3, and pSTAT3 (Tyr705) expression in HepG2 cells by knocking down or overexpressing LSINCT5. CCK-8– cell counting Kit-8; ceRNA – competing endogenous RNA; FISH – fluorescent in situ hybridization; HCC – hepatocellular carcinoma; LSINCT5 – long stress induced non-coding transcripts 5; miRNAs – micro RNAs; oe – overexpression; RIP – RNA immunoprecipitation.
Figure Legend Snippet: LSINCT5 acts as a ceRNA of miR-4516 to promote HCC progression. ( A ) RIP assays followed by qPCR to identify putative miRNAs associated with LSINCT5. ( B ) The miRNAs were verified by RNA pulldown a with biotin-labeled sense or antisense LSINCT5. ( C ) Luciferase reporter assays identified miR-4516 could interact with LSINCT5 (top). RNA pulldown assay with anti-AGO2 was performed in HepG2 cells with miR-4516 overexpression followed by qPCR to enrich LSINCT5 (bottom). ( D ) The predicted base-pairing between LSINCT5 and miR-4516 at miRDB. Three hits were detected. ( E ) RNA-FISH assay revealed the co-localization of LSINCT5 (Alexa Fluor 488, green dots) and miR-4516 (Cy3, red dots) in HepG2 cells. Scale bar, 5 μm. ( F ) CCK-8 viability assay for Huh7 cells and HepG2 cells transfected with plasmids as indicated. ( G ) Tumor sphere formation assay for HepG2 cells transfected with negative control (NC), miR-4516 mimics + oe Vec or miR-4516 mimics + LSINCT5. Quantification results were shown on the right panel. ( H ) Western blot assays for BclxL, STAT3, and pSTAT3 (Tyr705) expression in HepG2 cells by knocking down or overexpressing LSINCT5. CCK-8– cell counting Kit-8; ceRNA – competing endogenous RNA; FISH – fluorescent in situ hybridization; HCC – hepatocellular carcinoma; LSINCT5 – long stress induced non-coding transcripts 5; miRNAs – micro RNAs; oe – overexpression; RIP – RNA immunoprecipitation.

Techniques Used: Real-time Polymerase Chain Reaction, Labeling, Luciferase, Over Expression, Fluorescence In Situ Hybridization, CCK-8 Assay, Viability Assay, Transfection, Tube Formation Assay, Negative Control, Western Blot, Expressing, Cell Counting, In Situ Hybridization, Immunoprecipitation

17) Product Images from "LncRNA-SARCC suppresses renal cell carcinoma (RCC) progression via altering the androgen receptor(AR)/miRNA-143-3p signals"

Article Title: LncRNA-SARCC suppresses renal cell carcinoma (RCC) progression via altering the androgen receptor(AR)/miRNA-143-3p signals

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2017.74

LncRNA-SARCC is physically associated with and negatively correlated with AR. ( a ) Immunoblot of AR expression in a series of RCC cell lines or immortalized proximal tubule epithelial cell line from normal adult human kidney (HK2, A498, SW839, 769-P, ACHN, 786-O, OSRC-2, Caki-1 and Caki-2), with prostate cancer cell line C4-2 as positive control. ( b ) A schematic illustration of the procedure used to discover and define LncRNAs binding to AR in RCC tissues. ( c ) RIP assays for the potential LncRNA candidates endogenously associated with AR in SW839 cells. Total RNA was subjected to qRT-PCR assays. ( d ) Primary RCC and adjacent non-cancerous renal tissues were subjected to RNA FISH and analyzed by ultraviolet light excitation using a fluorescence microscope. ( e ) qRT-PCR for LncRNA-SARCC, GAPDH and U1 from RNA extracted from cytoplasmic and nuclear fractions. ( f ) SW839 and OSRC-2 cells were cross-linked with/without 4% paraformaldehyde before RIP assays were carried out. ( g ) RIP assay in 10 nM DHT-treated SW839 cells at the indicated time points. ( h ) RNAs corresponding to different fragments of LncRNA-SARCC were biotinylated and incubated with SW839 cell extracts, targeted with streptavidin beads and washed. Associated AR protein was detected by WB and compared between full length LncRNA-SARCC and its antisense RNA. ( i and j ) qRT-PCR assays (left panels) for the shRNA-SARCC mRNA level in stable SW839 ( i ) and OSRC-2 ( j ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( k and l ) qRT-PCR assays for the oe-SARCC mRNA levels in stable SW839 ( k ) and OSRC-2 ( l ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( m ) Immunofluorescence staining of AR. SW839-control and SW839-LncRNA-SARCC cells were hormone-starved for 3 days and the SW839-control cells treated with DMSO or 1 nM R1881, whereas the SW839-LncRNA-SARCC cells were treated with R1881 for 24 h before subjected to immunostaining using an anti-AR antibody. ( n ) SW839 cells expressing control shRNA or LncRNA-SARCC shRNA were treated with 20 mg/ml cycloheximide (CHX) for the indicated time periods and cell lysates analyzed by WB. ( o ) SW839 cells expressing mock or oe-LncRNA-SARCC were treated as in ( n ) and analyzed by WB. ( p ) SW839 cells expressing shRNA-control (−) or shRNA-SARCC (+) were cultured with/without 5 mM MG132 for 10 h and cell lysates analyzed by WB. ( q ) CoIP showing AR-Hsp90 protein interaction with the absence or presence of LncRNA-SARCC. AR, Hsp90 and LncRNA-SARCC were expressed in 293T cells through transient transfection followed by AR immunoprecipitation. AR and associated Hsp90 protein was detected by immunoblot analysis. Data shown are mean±S.D. ( n =3). * P
Figure Legend Snippet: LncRNA-SARCC is physically associated with and negatively correlated with AR. ( a ) Immunoblot of AR expression in a series of RCC cell lines or immortalized proximal tubule epithelial cell line from normal adult human kidney (HK2, A498, SW839, 769-P, ACHN, 786-O, OSRC-2, Caki-1 and Caki-2), with prostate cancer cell line C4-2 as positive control. ( b ) A schematic illustration of the procedure used to discover and define LncRNAs binding to AR in RCC tissues. ( c ) RIP assays for the potential LncRNA candidates endogenously associated with AR in SW839 cells. Total RNA was subjected to qRT-PCR assays. ( d ) Primary RCC and adjacent non-cancerous renal tissues were subjected to RNA FISH and analyzed by ultraviolet light excitation using a fluorescence microscope. ( e ) qRT-PCR for LncRNA-SARCC, GAPDH and U1 from RNA extracted from cytoplasmic and nuclear fractions. ( f ) SW839 and OSRC-2 cells were cross-linked with/without 4% paraformaldehyde before RIP assays were carried out. ( g ) RIP assay in 10 nM DHT-treated SW839 cells at the indicated time points. ( h ) RNAs corresponding to different fragments of LncRNA-SARCC were biotinylated and incubated with SW839 cell extracts, targeted with streptavidin beads and washed. Associated AR protein was detected by WB and compared between full length LncRNA-SARCC and its antisense RNA. ( i and j ) qRT-PCR assays (left panels) for the shRNA-SARCC mRNA level in stable SW839 ( i ) and OSRC-2 ( j ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( k and l ) qRT-PCR assays for the oe-SARCC mRNA levels in stable SW839 ( k ) and OSRC-2 ( l ) cell clones. AR protein and luciferase level were measured by WB (middle panels) and luciferase reporter assay (right panels). ( m ) Immunofluorescence staining of AR. SW839-control and SW839-LncRNA-SARCC cells were hormone-starved for 3 days and the SW839-control cells treated with DMSO or 1 nM R1881, whereas the SW839-LncRNA-SARCC cells were treated with R1881 for 24 h before subjected to immunostaining using an anti-AR antibody. ( n ) SW839 cells expressing control shRNA or LncRNA-SARCC shRNA were treated with 20 mg/ml cycloheximide (CHX) for the indicated time periods and cell lysates analyzed by WB. ( o ) SW839 cells expressing mock or oe-LncRNA-SARCC were treated as in ( n ) and analyzed by WB. ( p ) SW839 cells expressing shRNA-control (−) or shRNA-SARCC (+) were cultured with/without 5 mM MG132 for 10 h and cell lysates analyzed by WB. ( q ) CoIP showing AR-Hsp90 protein interaction with the absence or presence of LncRNA-SARCC. AR, Hsp90 and LncRNA-SARCC were expressed in 293T cells through transient transfection followed by AR immunoprecipitation. AR and associated Hsp90 protein was detected by immunoblot analysis. Data shown are mean±S.D. ( n =3). * P

Techniques Used: Expressing, Positive Control, Binding Assay, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Fluorescence, Microscopy, Incubation, Western Blot, shRNA, Clone Assay, Luciferase, Reporter Assay, Immunofluorescence, Staining, Immunostaining, Cell Culture, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation

18) Product Images from "HNF4A-AS1/hnRNPU/CTCF axis as a therapeutic target for aerobic glycolysis and neuroblastoma progression"

Article Title: HNF4A-AS1/hnRNPU/CTCF axis as a therapeutic target for aerobic glycolysis and neuroblastoma progression

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-020-00857-7

HNF4A-AS1 promotes aerobic glycolysis and NB progression. a Schematic illustration indicating genomic location of HNF4A-AS1 and HNF4A . Northern blot using a 254-bp specific probe and real-time qRT-PCR (normalized to β-actin, n = 5) showing the endogenous existence of HNF4A-AS1 transcript in normal dorsal root ganglia (DG), NB cell lines, and MCF 10A cells. b RNA-FISH using a 254-bp antisense probe showing localization (arrowheads) of HNF4A-AS1 in the nuclei (DAPI staining) of BE(2)-C cells, with sense probe and RNase A (20 μg) treatment as negative controls. Scale bars, 10 μm. c Real-time qRT-PCR (normalized to β-actin, n = 4) revealing the enrichment of HNF4A-AS1 in the cytoplasm and nuclei of NB cells. d Western blot assay indicating the expression of HNF4A and glycolytic genes in SH-SY5Y, SK-N-AS, and BE(2)-C cells stably transfected with empty vector (mock), HNF4A-AS1 , scramble shRNA (sh-Scb), or sh-HNF4A-AS1. e – g ECAR bars ( e ), soft agar ( f ), and matrigel invasion ( g ) assays showing glycolysis, anchor-independent growth, and invasion of NB cells stably transfected with mock, HNF4A-AS1 , sh-Scb, or sh-HNF4A-AS1, and those treated with 2-DG (10 mmol·L −1 , n = 4). h Representative images, in vivo growth curve, Ki-67 and CD31 immunostaining, and weight at the end points of subcutaneous xenograft tumors formed by SH-SY5Y cells stably transfected with mock or HNF4A-AS1 in nude mice ( n = 5 per group). Scale bar, 100 μm. i In vivo imaging, representative images and metastatic counts of lungs, and Kaplan-Meier curves of nude mice ( n = 5 per group) treated with tail vein injection of SH-SY5Y cells stably transfected with mock or HNF4A-AS1 . Scale bar, 100 μm. ANOVA and Student’s t test compared the difference in a and e – i . Log-rank test for survival comparison in i . * P
Figure Legend Snippet: HNF4A-AS1 promotes aerobic glycolysis and NB progression. a Schematic illustration indicating genomic location of HNF4A-AS1 and HNF4A . Northern blot using a 254-bp specific probe and real-time qRT-PCR (normalized to β-actin, n = 5) showing the endogenous existence of HNF4A-AS1 transcript in normal dorsal root ganglia (DG), NB cell lines, and MCF 10A cells. b RNA-FISH using a 254-bp antisense probe showing localization (arrowheads) of HNF4A-AS1 in the nuclei (DAPI staining) of BE(2)-C cells, with sense probe and RNase A (20 μg) treatment as negative controls. Scale bars, 10 μm. c Real-time qRT-PCR (normalized to β-actin, n = 4) revealing the enrichment of HNF4A-AS1 in the cytoplasm and nuclei of NB cells. d Western blot assay indicating the expression of HNF4A and glycolytic genes in SH-SY5Y, SK-N-AS, and BE(2)-C cells stably transfected with empty vector (mock), HNF4A-AS1 , scramble shRNA (sh-Scb), or sh-HNF4A-AS1. e – g ECAR bars ( e ), soft agar ( f ), and matrigel invasion ( g ) assays showing glycolysis, anchor-independent growth, and invasion of NB cells stably transfected with mock, HNF4A-AS1 , sh-Scb, or sh-HNF4A-AS1, and those treated with 2-DG (10 mmol·L −1 , n = 4). h Representative images, in vivo growth curve, Ki-67 and CD31 immunostaining, and weight at the end points of subcutaneous xenograft tumors formed by SH-SY5Y cells stably transfected with mock or HNF4A-AS1 in nude mice ( n = 5 per group). Scale bar, 100 μm. i In vivo imaging, representative images and metastatic counts of lungs, and Kaplan-Meier curves of nude mice ( n = 5 per group) treated with tail vein injection of SH-SY5Y cells stably transfected with mock or HNF4A-AS1 . Scale bar, 100 μm. ANOVA and Student’s t test compared the difference in a and e – i . Log-rank test for survival comparison in i . * P

Techniques Used: Northern Blot, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Staining, Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation, shRNA, In Vivo, Immunostaining, Mouse Assay, In Vivo Imaging, Injection

HNF4A-AS1 facilitates growth and invasion of NB cells via hnRNPU-mediated transactivation of CTCF. a Volcano plots of RNA-seq revealing alteration of gene expression (fold change > 2.0, P
Figure Legend Snippet: HNF4A-AS1 facilitates growth and invasion of NB cells via hnRNPU-mediated transactivation of CTCF. a Volcano plots of RNA-seq revealing alteration of gene expression (fold change > 2.0, P

Techniques Used: RNA Sequencing Assay, Expressing

HNF4A-AS1 interacts with hnRNPU protein in NB cells. a Coomassie blue staining (left panel) and mass spectrometry (MS) assay (right panel) of indicated electrophoretic bands revealing the identification of protein pulled down by biotin-labeled HNF4A-AS1 in BE(2)-C cells. b Biotin-labeled RNA pull-down and Western blot assays showing the hnRNPU protein pulled down by sense or antisense (AS) HNF4A-AS1 from lysates of BE(2)-C cells. The HNF4A-AS1 AS- and bead-bound protein served as negative controls. c Dual RNA-FISH and immunofluorescence staining assay indicating the co-localization of hnRNPU and HNF4A-AS1 in the nuclei of SH-SY5Y cells stably transfected with empty vector (mock) or HNF4A-AS1 . d RIP (upper right panel) and Western blot (lower right panel) assays using hnRNPU antibody showing the interaction between HNF4A-AS1 and hnRNPU protein in SH-SY5Y cells transfected with a series truncations of HNF4A-AS1 (left panel). The IgG-bound RNA was taken as a negative control. e Biotin-labeled RNA pull-down assay revealing the interaction between HNF4A-AS1 truncations and hnRNPU protein in BE(2)-C cells. Bead-bound protein served as a negative control. f RNA EMSA assay using biotin-labeled probes indicating the interaction of HNF4A-AS1 truncations with recombinant GST-tagged hnRNPU protein, with or without competition using an excess of unlabeled homologous probe. g Biotin-labeled RNA pull-down and Western blot assays showing the recovered hnRNPU truncations (upper panel) after incubation of biotin-labeled HNF4A-AS1 with full-length or truncated forms of GST-tagged recombinant hnRNPU protein (lower panel). h RIP assay using FLAG antibody indicating the interaction between HNF4A-AS1 truncations and FLAG-tagged hnRNPU protein in BE(2)-C cells. The IgG was applied as a negative control. Data are representative of three independent experiments in b – h
Figure Legend Snippet: HNF4A-AS1 interacts with hnRNPU protein in NB cells. a Coomassie blue staining (left panel) and mass spectrometry (MS) assay (right panel) of indicated electrophoretic bands revealing the identification of protein pulled down by biotin-labeled HNF4A-AS1 in BE(2)-C cells. b Biotin-labeled RNA pull-down and Western blot assays showing the hnRNPU protein pulled down by sense or antisense (AS) HNF4A-AS1 from lysates of BE(2)-C cells. The HNF4A-AS1 AS- and bead-bound protein served as negative controls. c Dual RNA-FISH and immunofluorescence staining assay indicating the co-localization of hnRNPU and HNF4A-AS1 in the nuclei of SH-SY5Y cells stably transfected with empty vector (mock) or HNF4A-AS1 . d RIP (upper right panel) and Western blot (lower right panel) assays using hnRNPU antibody showing the interaction between HNF4A-AS1 and hnRNPU protein in SH-SY5Y cells transfected with a series truncations of HNF4A-AS1 (left panel). The IgG-bound RNA was taken as a negative control. e Biotin-labeled RNA pull-down assay revealing the interaction between HNF4A-AS1 truncations and hnRNPU protein in BE(2)-C cells. Bead-bound protein served as a negative control. f RNA EMSA assay using biotin-labeled probes indicating the interaction of HNF4A-AS1 truncations with recombinant GST-tagged hnRNPU protein, with or without competition using an excess of unlabeled homologous probe. g Biotin-labeled RNA pull-down and Western blot assays showing the recovered hnRNPU truncations (upper panel) after incubation of biotin-labeled HNF4A-AS1 with full-length or truncated forms of GST-tagged recombinant hnRNPU protein (lower panel). h RIP assay using FLAG antibody indicating the interaction between HNF4A-AS1 truncations and FLAG-tagged hnRNPU protein in BE(2)-C cells. The IgG was applied as a negative control. Data are representative of three independent experiments in b – h

Techniques Used: Staining, Mass Spectrometry, Labeling, Western Blot, Fluorescence In Situ Hybridization, Immunofluorescence, Stable Transfection, Transfection, Plasmid Preparation, Negative Control, Pull Down Assay, Recombinant, Incubation

19) Product Images from "Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)"

Article Title: Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.005433

Degradation of mitochondrion-associated cytosolic rRNAs. A , comparison of total ER, mitochondrial ( Mito ) and cytosolic ( Cyto ) nucleic acids. The top panel on the left shows the nucleic acids on an EtBr agarose gel. Equal cell volume of mitochondria and ER were loaded. The bottom panels on the left display the immunoblots of ER, cytosolic, and mitochondrial markers. The middle panel shows the total mitochondrial RNAs with RNA markers on a denaturing gel. The top panel on the right displays nucleic acids in equal protein volume of ER and mitochondria. The bottom panel on the right displays the Coomassie staining of ER and mitochondrial lysates. nt , nucleotides. B , in organello decay of mitochondrion-associated cytosolic rRNAs. The top panel shows mtDNA and mitochondrion-associated cytosolic rRNAs in the mitochondrial pellets or the incubation buffer ( Sup ) at three time points. The middle panel shows the immunoblot of the samples. The bottom panel displays the quantification of the rRNAs ( n = 3). C , in organello decay of mitochondrion-associated cytosolic rRNAs and ER-associated rRNAs at pH 7.4 and pH 6.5. D , comparison of the decay of mitochondrion-associated cytosolic rRNAs ( Mito ) and rRNAs in the cytosol ( Cyto ). Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
Figure Legend Snippet: Degradation of mitochondrion-associated cytosolic rRNAs. A , comparison of total ER, mitochondrial ( Mito ) and cytosolic ( Cyto ) nucleic acids. The top panel on the left shows the nucleic acids on an EtBr agarose gel. Equal cell volume of mitochondria and ER were loaded. The bottom panels on the left display the immunoblots of ER, cytosolic, and mitochondrial markers. The middle panel shows the total mitochondrial RNAs with RNA markers on a denaturing gel. The top panel on the right displays nucleic acids in equal protein volume of ER and mitochondria. The bottom panel on the right displays the Coomassie staining of ER and mitochondrial lysates. nt , nucleotides. B , in organello decay of mitochondrion-associated cytosolic rRNAs. The top panel shows mtDNA and mitochondrion-associated cytosolic rRNAs in the mitochondrial pellets or the incubation buffer ( Sup ) at three time points. The middle panel shows the immunoblot of the samples. The bottom panel displays the quantification of the rRNAs ( n = 3). C , in organello decay of mitochondrion-associated cytosolic rRNAs and ER-associated rRNAs at pH 7.4 and pH 6.5. D , comparison of the decay of mitochondrion-associated cytosolic rRNAs ( Mito ) and rRNAs in the cytosol ( Cyto ). Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

Techniques Used: Agarose Gel Electrophoresis, Western Blot, Staining, Incubation

Mitochondrial RNASET2 regulates cytosolic rRNA levels and nuclear transcription in vivo . A , in vivo RNA synthesis in HEK and RNASET2-overexpressing cells ( T2 ). The lower panel on the left is an immunoblot of tubulin. The right panel shows the quantification of biotinylated RNAs. nt , nucleotides. B , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2-overexpressing ( T2 ) cells. C , in vivo RNA synthesis in HEK and RNASET2 knockdown cells ( KD ). D , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2 knockdown ( KD ) cells. E , in vivo RNA synthesis in HEK cells and cells expressing C1-ΔN-RNASET2 ( C1 Δ NT2 ). F , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK and C1-ΔN-RNASET2 expressing ( C1 Δ NT2 ) cells. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
Figure Legend Snippet: Mitochondrial RNASET2 regulates cytosolic rRNA levels and nuclear transcription in vivo . A , in vivo RNA synthesis in HEK and RNASET2-overexpressing cells ( T2 ). The lower panel on the left is an immunoblot of tubulin. The right panel shows the quantification of biotinylated RNAs. nt , nucleotides. B , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2-overexpressing ( T2 ) cells. C , in vivo RNA synthesis in HEK and RNASET2 knockdown cells ( KD ). D , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2 knockdown ( KD ) cells. E , in vivo RNA synthesis in HEK cells and cells expressing C1-ΔN-RNASET2 ( C1 Δ NT2 ). F , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK and C1-ΔN-RNASET2 expressing ( C1 Δ NT2 ) cells. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

Techniques Used: In Vivo, Quantitative RT-PCR, Expressing

Mitochondrion-associated cytosolic rRNAs are not degraded by a cytosolic nuclease. A , no RNase activity resides at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in MitoPrep buffer ( Isotonic ) or hypotonic buffer that ruptures the mitochondrial outer membrane. Biotinylated 28S r RNA was added to the mixture and incubated at 37 °C for 1 or 30 min before the reaction was terminated. The bottom panel shows the mortalin immunoblot of the samples. nt , nucleotides. B , immunoblotting of a mitochondrial IMS protein DDP2 and matrix protein mortalin in the in organello degradation mitochondrial samples at 0, 30, and 60 min and the 0-min sample in the hypotonic buffer ( Hypo ). C , purified mitochondrial IMS fraction was tested for nuclease activity with purified cytosolic RNAs as substrates. The bottom panel shows the immunoblot of mitochondrial IMS protein DDP2. D , in organello decay of mitochondrion-associated cytosolic rRNAs with or without the addition of purified mitochondrial IMS fraction. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
Figure Legend Snippet: Mitochondrion-associated cytosolic rRNAs are not degraded by a cytosolic nuclease. A , no RNase activity resides at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in MitoPrep buffer ( Isotonic ) or hypotonic buffer that ruptures the mitochondrial outer membrane. Biotinylated 28S r RNA was added to the mixture and incubated at 37 °C for 1 or 30 min before the reaction was terminated. The bottom panel shows the mortalin immunoblot of the samples. nt , nucleotides. B , immunoblotting of a mitochondrial IMS protein DDP2 and matrix protein mortalin in the in organello degradation mitochondrial samples at 0, 30, and 60 min and the 0-min sample in the hypotonic buffer ( Hypo ). C , purified mitochondrial IMS fraction was tested for nuclease activity with purified cytosolic RNAs as substrates. The bottom panel shows the immunoblot of mitochondrial IMS protein DDP2. D , in organello decay of mitochondrion-associated cytosolic rRNAs with or without the addition of purified mitochondrial IMS fraction. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

Techniques Used: Activity Assay, Isolation, Incubation, Purification

Cytosolic rRNAs are transported to the IMS RNA degradation machinery through the mitochondrial RNA import pathway. A , immunoblots of HEK and PNPASE-overexpressing ( PNP ) cell lysates. Mortalin was used as a loading control. B , in organello degradation of mitochondrion-associated 28S and 18S rRNAs on isolated control mitochondria ( HEK ) and mitochondria overexpressing PNPASE ( PNP ) with 2 m m ATP. The grafts show quantification of 28S rRNAs. C , immunoblots of TM6 and PNPASE knockdown TM6 ( Cre ) cell lysates. Mortalin was used as a loading control. D , in organello degradation of mitochondrion-associated 28S and 18S rRNAs on isolated control mitochondria ( TM6 ) and PNPASE knockdown mitochondria ( Cre ). The grafts show quantification of 28S rRNAs. E , immunoblots of the lysates of HEK cells harboring an empty vector ( HEK ), overexpressing VDAC1, or expressing VDAC1 with a His Tag (VDAC1-His). Mortalin was used as a loading control. F , in organello degradation of mitochondrion-associated 28S and 18S rRNAs on isolated control mitochondria ( HEK ) and mitochondria overexpressing VDAC1 ( VDAC1 ). The grafts show quantification of 28S rRNAs. G , in organello degradation of mitochondrion-associated 28S and 18S rRNAs on isolated control mitochondria ( HEK ) and mitochondria overexpressing VDAC1 with a His tag ( VDAC1-His ). The grafts show quantification of 28S rRNAs. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
Figure Legend Snippet: Cytosolic rRNAs are transported to the IMS RNA degradation machinery through the mitochondrial RNA import pathway. A , immunoblots of HEK and PNPASE-overexpressing ( PNP ) cell lysates. Mortalin was used as a loading control. B , in organello degradation of mitochondrion-associated 28S and 18S rRNAs on isolated control mitochondria ( HEK ) and mitochondria overexpressing PNPASE ( PNP ) with 2 m m ATP. The grafts show quantification of 28S rRNAs. C , immunoblots of TM6 and PNPASE knockdown TM6 ( Cre ) cell lysates. Mortalin was used as a loading control. D , in organello degradation of mitochondrion-associated 28S and 18S rRNAs on isolated control mitochondria ( TM6 ) and PNPASE knockdown mitochondria ( Cre ). The grafts show quantification of 28S rRNAs. E , immunoblots of the lysates of HEK cells harboring an empty vector ( HEK ), overexpressing VDAC1, or expressing VDAC1 with a His Tag (VDAC1-His). Mortalin was used as a loading control. F , in organello degradation of mitochondrion-associated 28S and 18S rRNAs on isolated control mitochondria ( HEK ) and mitochondria overexpressing VDAC1 ( VDAC1 ). The grafts show quantification of 28S rRNAs. G , in organello degradation of mitochondrion-associated 28S and 18S rRNAs on isolated control mitochondria ( HEK ) and mitochondria overexpressing VDAC1 with a His tag ( VDAC1-His ). The grafts show quantification of 28S rRNAs. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

Techniques Used: Western Blot, Isolation, Plasmid Preparation, Expressing

20) Product Images from "The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma"

Article Title: The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma

Journal: Nature Communications

doi: 10.1038/s41467-018-04006-0

The association between LINC01138 and PRMT5 is a candidate therapeutic target for HCC. a RNA pull-down assays for the specific association of PRMT5 and LINC01138, in the cells exposed to PRMT5 inhibitors at the indicated concentration for 48 h. b SMMC-7721 cells were transfected with plasmids containing HA-tagged PRMT5, and RIP assays were performed using HA antibodies in the cells treated with PRMT5 inhibitors at the indicated concentration for 48 h. c CCK-8 assays for pWPXL-LINC01138 or vector cells, exposed to PRMT5 inhibitors at the indicated concentration for 3 days. d CCK-8 assays in cells transfected with si-NC or si-LINC01138 mixture, exposed to PRMT5 inhibitors at the indicated concentration for 3 days. e IC 50 were analysed in cells transfected with si-NC or si-LINC01138 mixture, exposed to PRMT5 inhibitors for 3 days. Values are expressed as the mean ± SEM, n = 3 b – e . ** P
Figure Legend Snippet: The association between LINC01138 and PRMT5 is a candidate therapeutic target for HCC. a RNA pull-down assays for the specific association of PRMT5 and LINC01138, in the cells exposed to PRMT5 inhibitors at the indicated concentration for 48 h. b SMMC-7721 cells were transfected with plasmids containing HA-tagged PRMT5, and RIP assays were performed using HA antibodies in the cells treated with PRMT5 inhibitors at the indicated concentration for 48 h. c CCK-8 assays for pWPXL-LINC01138 or vector cells, exposed to PRMT5 inhibitors at the indicated concentration for 3 days. d CCK-8 assays in cells transfected with si-NC or si-LINC01138 mixture, exposed to PRMT5 inhibitors at the indicated concentration for 3 days. e IC 50 were analysed in cells transfected with si-NC or si-LINC01138 mixture, exposed to PRMT5 inhibitors for 3 days. Values are expressed as the mean ± SEM, n = 3 b – e . ** P

Techniques Used: Concentration Assay, Transfection, CCK-8 Assay, Plasmid Preparation

The oncogenic IGF2BP1/IGF2BP3-LINC01138-PRMT5 axis in HCC cells. a Relative RNA levels of LINC01138 in SMMC-7721 cells with IGF2BP1/IGF2BP3 knockdown or overexpression, using qPCR. b The half-life of LINC01138 after treatment with 2.5 μM actinomycin D for indicated times, with IGF2BP1/IGF2BP3 knockdown or overexpression in SMMC-7721 cells. c CCK-8 rescue assays were performed after LINC01138 knockdown in pWPXL-IGF2BP1 or pWPXL-IGF2BP3 cells. d Transwell invasion rescue assays were performed after LINC01138 knockdown in pWPXL-IGF2BP1 or pWPXL-IGF2BP3 cells. e Immunoblotting to detect the protein levels of PRMT5 after knockdown of IGF2BP1/IGF2BP3 or LINC01138 in SMMC-7721 cells. f Integrated model depicting lncRNA LINC01138 as an oncogene in liver cancer. The working model shows that LINC01138 loci is amplified, and its transcript is stabilized by IGF2BP1 or IGF2BP3 in HCC; LINC01138 exerts its oncogenic activity through interacting with and stabilizing PRMT5, which can be disrupted by small molecule inhibitors in HCC. Values are expressed as the mean ± SEM, n = 3 in a – d . *** P
Figure Legend Snippet: The oncogenic IGF2BP1/IGF2BP3-LINC01138-PRMT5 axis in HCC cells. a Relative RNA levels of LINC01138 in SMMC-7721 cells with IGF2BP1/IGF2BP3 knockdown or overexpression, using qPCR. b The half-life of LINC01138 after treatment with 2.5 μM actinomycin D for indicated times, with IGF2BP1/IGF2BP3 knockdown or overexpression in SMMC-7721 cells. c CCK-8 rescue assays were performed after LINC01138 knockdown in pWPXL-IGF2BP1 or pWPXL-IGF2BP3 cells. d Transwell invasion rescue assays were performed after LINC01138 knockdown in pWPXL-IGF2BP1 or pWPXL-IGF2BP3 cells. e Immunoblotting to detect the protein levels of PRMT5 after knockdown of IGF2BP1/IGF2BP3 or LINC01138 in SMMC-7721 cells. f Integrated model depicting lncRNA LINC01138 as an oncogene in liver cancer. The working model shows that LINC01138 loci is amplified, and its transcript is stabilized by IGF2BP1 or IGF2BP3 in HCC; LINC01138 exerts its oncogenic activity through interacting with and stabilizing PRMT5, which can be disrupted by small molecule inhibitors in HCC. Values are expressed as the mean ± SEM, n = 3 in a – d . *** P

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, CCK-8 Assay, Amplification, Activity Assay

LINC01138 is associated with clinical outcomes in patients with HCC. a The flow chart for selecting candidate lincRNAs in HCC; four lincRNAs were identified for further study. b The copy numbers of LINC01138 were determined in 72 pairs of HCC tissues and adjacent normal tissues using qPCR. c Fold-change of LINC01138 copy number variations in 72 paired tissues (deletion, blue; no-change, yellow; amplification, red.). d The RNA levels of LINC01138 were quantified in 120 pairs of HCC tissues and adjacent normal tissues using qPCR. e Fold-changes of expression of LINC01138 in 120 paired tissues (downexpression, blue; no-change, yellow; upexpression, red.). f Clinical significance of LINC01138 in patients with HCC; high LINC01138 expression positively correlated with tumour size (≥5 cm), AFP ( > 200 ng/ml) and HBsAg-positive patients. g Kaplan–Meier analyses of the correlation between LINC01138 RNA levels and the overall survival in 120 patients with HCC. Patients were stratified for the analysis by the median value. Values are expressed as the median with interquartile range in b , d , f
Figure Legend Snippet: LINC01138 is associated with clinical outcomes in patients with HCC. a The flow chart for selecting candidate lincRNAs in HCC; four lincRNAs were identified for further study. b The copy numbers of LINC01138 were determined in 72 pairs of HCC tissues and adjacent normal tissues using qPCR. c Fold-change of LINC01138 copy number variations in 72 paired tissues (deletion, blue; no-change, yellow; amplification, red.). d The RNA levels of LINC01138 were quantified in 120 pairs of HCC tissues and adjacent normal tissues using qPCR. e Fold-changes of expression of LINC01138 in 120 paired tissues (downexpression, blue; no-change, yellow; upexpression, red.). f Clinical significance of LINC01138 in patients with HCC; high LINC01138 expression positively correlated with tumour size (≥5 cm), AFP ( > 200 ng/ml) and HBsAg-positive patients. g Kaplan–Meier analyses of the correlation between LINC01138 RNA levels and the overall survival in 120 patients with HCC. Patients were stratified for the analysis by the median value. Values are expressed as the median with interquartile range in b , d , f

Techniques Used: Flow Cytometry, Real-time Polymerase Chain Reaction, Amplification, Expressing

LINC01138 physically interacts with IGF2BP1, IGF2BP3 and PRMT5 in HCC cells. a Immunoblotting for the specific associations of IGF2BP1, IGF2BP3 or PRMT5 with biotinylated-LINC01138 from three independent streptavidin RNA pull-down assays. b RIP assays were performed using the indicated antibodies. Real-time PCR was used to detect LINC01138 enrichment, using GAPDH antibody as the antibody control and 2036-nt PVT1 as the LincRNA control. c Immunoblotting of IGF2BP1, IGF2BP3 or PRMT5 in pull-down samples by full-length biotinylated-LINC01138 (#1) or truncated biotinylated-LINC01138 RNA motifs (#2: 1–219 nt; #3: 1–630 nt; #4: 1–1560 nt; #5: 630–2075 nt; #6: 1560–2075 nt; #7: 220–2075 nt), with GAPDH as the negative control. d – f Deletion mapping for the domains of IGF2BP1 ( d ), IGF2BP3 ( e ) or PRMT5 ( f ) that bind to LINC01138. RIP analysis for LINC01138 enrichment in cells transiently transfected with plasmids containing the indicated FLAG-tagged or HA-tagged full-length or truncated constructs. Values are expressed as the mean ± SEM, n = 3 in b , d – f
Figure Legend Snippet: LINC01138 physically interacts with IGF2BP1, IGF2BP3 and PRMT5 in HCC cells. a Immunoblotting for the specific associations of IGF2BP1, IGF2BP3 or PRMT5 with biotinylated-LINC01138 from three independent streptavidin RNA pull-down assays. b RIP assays were performed using the indicated antibodies. Real-time PCR was used to detect LINC01138 enrichment, using GAPDH antibody as the antibody control and 2036-nt PVT1 as the LincRNA control. c Immunoblotting of IGF2BP1, IGF2BP3 or PRMT5 in pull-down samples by full-length biotinylated-LINC01138 (#1) or truncated biotinylated-LINC01138 RNA motifs (#2: 1–219 nt; #3: 1–630 nt; #4: 1–1560 nt; #5: 630–2075 nt; #6: 1560–2075 nt; #7: 220–2075 nt), with GAPDH as the negative control. d – f Deletion mapping for the domains of IGF2BP1 ( d ), IGF2BP3 ( e ) or PRMT5 ( f ) that bind to LINC01138. RIP analysis for LINC01138 enrichment in cells transiently transfected with plasmids containing the indicated FLAG-tagged or HA-tagged full-length or truncated constructs. Values are expressed as the mean ± SEM, n = 3 in b , d – f

Techniques Used: Real-time Polymerase Chain Reaction, Negative Control, Transfection, Construct

21) Product Images from "The Post-transcriptional Regulator rsmA/csrA Activates T3SS by Stabilizing the 5? UTR of hrpG, the Master Regulator of hrp/hrc Genes, in Xanthomonas"

Article Title: The Post-transcriptional Regulator rsmA/csrA Activates T3SS by Stabilizing the 5? UTR of hrpG, the Master Regulator of hrp/hrc Genes, in Xanthomonas

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003945

RNA mobility shift assays with purified 6HisRsmA of X. citri subsp. citri A ) 6HisRsmAxcc (65 nM) binds to the high affinity RNA target R9-43. Biotin 3′-end-labeled R9-43 (6.25 nM) was incubated with 6HisRsmAxcc (65 nM) for 30 minutes at room temperature, followed by analysis on a 5% native polyacrylamide gel. A competitive assay in which unlabeled R9-43 RNA (6.25 nM) was added to the reaction reduced the signal resulting from the biotinylated nucleotide. B ) 6HisRsmAxcc directly interacts with the 5′ UTRs of hrpG and hrpD . The leader sequences of hrpD , hrpE and hrpG cloned were transcribed in vitro and biotinylated with RNA Labeling kit (Roche). Biotinylated RNA probes were incubated with 6HisRsmAxcc and resolved in a 5% native polyacrylamide gel. The addition of unlabeled competitor R9-43 to the reactions reduced the intensity of the shifted band, which confirmed the specificity of the RsmAxcc- hrpG and RsmAxcc- hrpD interactions. C ) 3′-end-biotin-labeled RNA probes encoding the leader sequences of hrpB , hrpC , hrpF and hrpX were tested for interactions with 6HisRsmAxcc ( Table S4 ). In addition, 3′-end-biotin–labeled RNA probes hrpG1 and hrpG2 , which bear the GGA motifs encoded by the 5′ leader sequence of hrpG , were used to map the interaction RsmAxcc- hrpG . Only the GGA motif between nucleotides 80 and 120 in the hrpG leader sequence ( hrpG 2 probe) interacted with 6HisRsmAxcc. D ) To determinate the apparent equilibrium binding constant (K d ), 3′ end-labeled hrpG 2 RNA (6.25 nM) was incubated with increasing concentrations of 6HisRsmAxcc as noted at the bottom of each lane. The binding curve for the 6HisRsmAxcc- hrpG 2 interaction was determined as a function of 6HisRsmAxcc concentration and shifted band intensity. The average pixel value of each shifted band was calculated with ImageJ software [68] , [69] , [112] . The apparent equilibrium binding constant (K d ) for this reaction was 0.18±0.2 µM 6HisRsmAxcc. Samples were loaded and resolved onto a 5% native polyacrylamide gel. All probes were transferred and cross-linked to a nylon membrane, incubated with streptavidin conjugated with horseradish peroxidase, and detected according to manufacturer's instructions (LightShiftChemiluminescent RNA EMSA Kit, Thermo Scientific). Signals + and − correspond to the presence and absence in the reaction, respectively. Positions of bound and free probes are shown.
Figure Legend Snippet: RNA mobility shift assays with purified 6HisRsmA of X. citri subsp. citri A ) 6HisRsmAxcc (65 nM) binds to the high affinity RNA target R9-43. Biotin 3′-end-labeled R9-43 (6.25 nM) was incubated with 6HisRsmAxcc (65 nM) for 30 minutes at room temperature, followed by analysis on a 5% native polyacrylamide gel. A competitive assay in which unlabeled R9-43 RNA (6.25 nM) was added to the reaction reduced the signal resulting from the biotinylated nucleotide. B ) 6HisRsmAxcc directly interacts with the 5′ UTRs of hrpG and hrpD . The leader sequences of hrpD , hrpE and hrpG cloned were transcribed in vitro and biotinylated with RNA Labeling kit (Roche). Biotinylated RNA probes were incubated with 6HisRsmAxcc and resolved in a 5% native polyacrylamide gel. The addition of unlabeled competitor R9-43 to the reactions reduced the intensity of the shifted band, which confirmed the specificity of the RsmAxcc- hrpG and RsmAxcc- hrpD interactions. C ) 3′-end-biotin-labeled RNA probes encoding the leader sequences of hrpB , hrpC , hrpF and hrpX were tested for interactions with 6HisRsmAxcc ( Table S4 ). In addition, 3′-end-biotin–labeled RNA probes hrpG1 and hrpG2 , which bear the GGA motifs encoded by the 5′ leader sequence of hrpG , were used to map the interaction RsmAxcc- hrpG . Only the GGA motif between nucleotides 80 and 120 in the hrpG leader sequence ( hrpG 2 probe) interacted with 6HisRsmAxcc. D ) To determinate the apparent equilibrium binding constant (K d ), 3′ end-labeled hrpG 2 RNA (6.25 nM) was incubated with increasing concentrations of 6HisRsmAxcc as noted at the bottom of each lane. The binding curve for the 6HisRsmAxcc- hrpG 2 interaction was determined as a function of 6HisRsmAxcc concentration and shifted band intensity. The average pixel value of each shifted band was calculated with ImageJ software [68] , [69] , [112] . The apparent equilibrium binding constant (K d ) for this reaction was 0.18±0.2 µM 6HisRsmAxcc. Samples were loaded and resolved onto a 5% native polyacrylamide gel. All probes were transferred and cross-linked to a nylon membrane, incubated with streptavidin conjugated with horseradish peroxidase, and detected according to manufacturer's instructions (LightShiftChemiluminescent RNA EMSA Kit, Thermo Scientific). Signals + and − correspond to the presence and absence in the reaction, respectively. Positions of bound and free probes are shown.

Techniques Used: Mobility Shift, Purification, Labeling, Incubation, Clone Assay, In Vitro, Sequencing, Binding Assay, Concentration Assay, Software

22) Product Images from "Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)"

Article Title: Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.005433

Degradation of mitochondrion-associated cytosolic rRNAs. A , comparison of total ER, mitochondrial ( Mito ) and cytosolic ( Cyto ) nucleic acids. The top panel on the left shows the nucleic acids on an EtBr agarose gel. Equal cell volume of mitochondria and ER were loaded. The bottom panels on the left display the immunoblots of ER, cytosolic, and mitochondrial markers. The middle panel shows the total mitochondrial RNAs with RNA markers on a denaturing gel. The top panel on the right displays nucleic acids in equal protein volume of ER and mitochondria. The bottom panel on the right displays the Coomassie staining of ER and mitochondrial lysates. nt , nucleotides. B , in organello decay of mitochondrion-associated cytosolic rRNAs. The top panel shows mtDNA and mitochondrion-associated cytosolic rRNAs in the mitochondrial pellets or the incubation buffer ( Sup ) at three time points. The middle panel shows the immunoblot of the samples. The bottom panel displays the quantification of the rRNAs ( n = 3). C , in organello decay of mitochondrion-associated cytosolic rRNAs and ER-associated rRNAs at pH 7.4 and pH 6.5. D , comparison of the decay of mitochondrion-associated cytosolic rRNAs ( Mito ) and rRNAs in the cytosol ( Cyto ). Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
Figure Legend Snippet: Degradation of mitochondrion-associated cytosolic rRNAs. A , comparison of total ER, mitochondrial ( Mito ) and cytosolic ( Cyto ) nucleic acids. The top panel on the left shows the nucleic acids on an EtBr agarose gel. Equal cell volume of mitochondria and ER were loaded. The bottom panels on the left display the immunoblots of ER, cytosolic, and mitochondrial markers. The middle panel shows the total mitochondrial RNAs with RNA markers on a denaturing gel. The top panel on the right displays nucleic acids in equal protein volume of ER and mitochondria. The bottom panel on the right displays the Coomassie staining of ER and mitochondrial lysates. nt , nucleotides. B , in organello decay of mitochondrion-associated cytosolic rRNAs. The top panel shows mtDNA and mitochondrion-associated cytosolic rRNAs in the mitochondrial pellets or the incubation buffer ( Sup ) at three time points. The middle panel shows the immunoblot of the samples. The bottom panel displays the quantification of the rRNAs ( n = 3). C , in organello decay of mitochondrion-associated cytosolic rRNAs and ER-associated rRNAs at pH 7.4 and pH 6.5. D , comparison of the decay of mitochondrion-associated cytosolic rRNAs ( Mito ) and rRNAs in the cytosol ( Cyto ). Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

Techniques Used: Agarose Gel Electrophoresis, Western Blot, Staining, Incubation

Mitochondrial RNASET2 regulates cytosolic rRNA levels and nuclear transcription in vivo . A , in vivo RNA synthesis in HEK and RNASET2-overexpressing cells ( T2 ). The lower panel on the left is an immunoblot of tubulin. The right panel shows the quantification of biotinylated RNAs. nt , nucleotides. B , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2-overexpressing ( T2 ) cells. C , in vivo RNA synthesis in HEK and RNASET2 knockdown cells ( KD ). D , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2 knockdown ( KD ) cells. E , in vivo RNA synthesis in HEK cells and cells expressing C1-ΔN-RNASET2 ( C1 Δ NT2 ). F , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK and C1-ΔN-RNASET2 expressing ( C1 Δ NT2 ) cells. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
Figure Legend Snippet: Mitochondrial RNASET2 regulates cytosolic rRNA levels and nuclear transcription in vivo . A , in vivo RNA synthesis in HEK and RNASET2-overexpressing cells ( T2 ). The lower panel on the left is an immunoblot of tubulin. The right panel shows the quantification of biotinylated RNAs. nt , nucleotides. B , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2-overexpressing ( T2 ) cells. C , in vivo RNA synthesis in HEK and RNASET2 knockdown cells ( KD ). D , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2 knockdown ( KD ) cells. E , in vivo RNA synthesis in HEK cells and cells expressing C1-ΔN-RNASET2 ( C1 Δ NT2 ). F , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK and C1-ΔN-RNASET2 expressing ( C1 Δ NT2 ) cells. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

Techniques Used: In Vivo, Quantitative RT-PCR, Expressing

Mitochondrion-associated cytosolic rRNAs are not degraded by a cytosolic nuclease. A , no RNase activity resides at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in MitoPrep buffer ( Isotonic ) or hypotonic buffer that ruptures the mitochondrial outer membrane. Biotinylated 28S r RNA was added to the mixture and incubated at 37 °C for 1 or 30 min before the reaction was terminated. The bottom panel shows the mortalin immunoblot of the samples. nt , nucleotides. B , immunoblotting of a mitochondrial IMS protein DDP2 and matrix protein mortalin in the in organello degradation mitochondrial samples at 0, 30, and 60 min and the 0-min sample in the hypotonic buffer ( Hypo ). C , purified mitochondrial IMS fraction was tested for nuclease activity with purified cytosolic RNAs as substrates. The bottom panel shows the immunoblot of mitochondrial IMS protein DDP2. D , in organello decay of mitochondrion-associated cytosolic rRNAs with or without the addition of purified mitochondrial IMS fraction. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
Figure Legend Snippet: Mitochondrion-associated cytosolic rRNAs are not degraded by a cytosolic nuclease. A , no RNase activity resides at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in MitoPrep buffer ( Isotonic ) or hypotonic buffer that ruptures the mitochondrial outer membrane. Biotinylated 28S r RNA was added to the mixture and incubated at 37 °C for 1 or 30 min before the reaction was terminated. The bottom panel shows the mortalin immunoblot of the samples. nt , nucleotides. B , immunoblotting of a mitochondrial IMS protein DDP2 and matrix protein mortalin in the in organello degradation mitochondrial samples at 0, 30, and 60 min and the 0-min sample in the hypotonic buffer ( Hypo ). C , purified mitochondrial IMS fraction was tested for nuclease activity with purified cytosolic RNAs as substrates. The bottom panel shows the immunoblot of mitochondrial IMS protein DDP2. D , in organello decay of mitochondrion-associated cytosolic rRNAs with or without the addition of purified mitochondrial IMS fraction. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

Techniques Used: Activity Assay, Isolation, Incubation, Purification

23) Product Images from "Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL"

Article Title: Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

doi: 10.4143/crt.2017.226

Bcl-xL mRNA level is positively correlated with long noncoding RNA HEIH (lncRNA-HEIH) expression level in colorectal cancer (CRC) tissues. (A) Bcl-xL mRNA levels in 84 paired CRC and adjacent normal mucosa were detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. ***p
Figure Legend Snippet: Bcl-xL mRNA level is positively correlated with long noncoding RNA HEIH (lncRNA-HEIH) expression level in colorectal cancer (CRC) tissues. (A) Bcl-xL mRNA levels in 84 paired CRC and adjacent normal mucosa were detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. ***p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Long noncoding RNA HEIH (lncRNA-HEIH) counteracts miR-939‒mediated transcriptional repression of Bcl-xL. (A) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, chromatin immunoprecipitation (ChIP) assays with nuclear factor κB (NF-κB) specific antibody were performed, and the retrieved DNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to input. (B) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, ChIP assays with NF-κB specific antibody were performed, and the retrieved DNA was detected by qRT-PCR and normalized to input. (C) After co-transfection of lncRNA-HEIH or lncRNA-HEIH-mut and luciferase reporter containing Bcl-xL promoterinto HT-29 cells, the luciferase activities were measured. Results are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. (D) After co-transfection of lncRNA-HEIH knockdown plasmid, miR-939 inhibitors, and luciferase reporter containing Bcl-xL promoter into LoVo cells, the luciferase activities were measured. Results are shown as in panel C. (E) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to GAPDH. (G) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. (H) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. Results are shown as mean±standard deviation from three independent experiments. **p
Figure Legend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) counteracts miR-939‒mediated transcriptional repression of Bcl-xL. (A) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, chromatin immunoprecipitation (ChIP) assays with nuclear factor κB (NF-κB) specific antibody were performed, and the retrieved DNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to input. (B) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, ChIP assays with NF-κB specific antibody were performed, and the retrieved DNA was detected by qRT-PCR and normalized to input. (C) After co-transfection of lncRNA-HEIH or lncRNA-HEIH-mut and luciferase reporter containing Bcl-xL promoterinto HT-29 cells, the luciferase activities were measured. Results are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. (D) After co-transfection of lncRNA-HEIH knockdown plasmid, miR-939 inhibitors, and luciferase reporter containing Bcl-xL promoter into LoVo cells, the luciferase activities were measured. Results are shown as in panel C. (E) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to GAPDH. (G) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. (H) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. Results are shown as mean±standard deviation from three independent experiments. **p

Techniques Used: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cotransfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Standard Deviation

Long noncoding RNA HEIH (lncRNA-HEIH) physically binds to miR-939 and counteracts the binding between miR-939 and nuclear factor κB (NF-κB). (A) Schematic outlining the predicted miR-939 binding sites on lncRNA-HEIH. The red nucleotides indict the seed sequences of miR-939. (B) HT-29 cell lysates were incubated with biotinylated lncRNA-HEIH or the miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut); after pull-down, RNAs were retrieved and detected by quantitative real-time polymerase chain reaction (qRT-PCR), and normalized to input. (C) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, RNA immunoprecipitation assay (RIP) assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. (D) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, RIP assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. Results are shown as mean±standard deviation from three independent experiments. **p
Figure Legend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) physically binds to miR-939 and counteracts the binding between miR-939 and nuclear factor κB (NF-κB). (A) Schematic outlining the predicted miR-939 binding sites on lncRNA-HEIH. The red nucleotides indict the seed sequences of miR-939. (B) HT-29 cell lysates were incubated with biotinylated lncRNA-HEIH or the miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut); after pull-down, RNAs were retrieved and detected by quantitative real-time polymerase chain reaction (qRT-PCR), and normalized to input. (C) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, RNA immunoprecipitation assay (RIP) assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. (D) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, RIP assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. Results are shown as mean±standard deviation from three independent experiments. **p

Techniques Used: Binding Assay, Incubation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Immunoprecipitation, Cotransfection, Plasmid Preparation, Standard Deviation

Long noncoding RNA HEIH (lncRNA-HEIH) is up-regulated in colorectal cancer (CRC) and indicts poor prognosis of CRC patients. (A) The expression of lncRNA-HEIH in 84 paired CRC and adjacent normal mucosa was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ***p
Figure Legend Snippet: Long noncoding RNA HEIH (lncRNA-HEIH) is up-regulated in colorectal cancer (CRC) and indicts poor prognosis of CRC patients. (A) The expression of lncRNA-HEIH in 84 paired CRC and adjacent normal mucosa was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ***p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

The mutation of miR-939 binding sites on long noncoding RNA HEIH (lncRNA-HEIH) abolished the effects of lncRNA-HEIH on colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. **p
Figure Legend Snippet: The mutation of miR-939 binding sites on long noncoding RNA HEIH (lncRNA-HEIH) abolished the effects of lncRNA-HEIH on colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. **p

Techniques Used: Mutagenesis, Binding Assay, Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

Knockdown of long noncoding RNA HEIH (lncRNA-HEIH) inhibits colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably knocked-down and control LoVo cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably knocked-down and control LoVo were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. *p
Figure Legend Snippet: Knockdown of long noncoding RNA HEIH (lncRNA-HEIH) inhibits colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably knocked-down and control LoVo cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably knocked-down and control LoVo were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. *p

Techniques Used: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

Enhanced expression of long noncoding RNA HEIH (lncRNA-HEIH) promotes colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphat e dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD). from three independent experiments. **p
Figure Legend Snippet: Enhanced expression of long noncoding RNA HEIH (lncRNA-HEIH) promotes colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphat e dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD). from three independent experiments. **p

Techniques Used: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, Cell Counting, Labeling, End Labeling, TUNEL Assay, Staining, Standard Deviation

24) Product Images from "Hypoxia-induced long non-coding RNA DARS-AS1 regulates RBM39 stability to promote myeloma malignancy"

Article Title: Hypoxia-induced long non-coding RNA DARS-AS1 regulates RBM39 stability to promote myeloma malignancy

Journal: Haematologica

doi: 10.3324/haematol.2019.218289

HIF-1 directly upregulates the expression of DARS-AS1, which may, in turn, enhance the expression of HIF-1α. (A) HIF-1α or HIF-2α was knocked down or overexpressed in 293T cells. DARS-AS1 expression levels were determined by quantitative polymerase chain reaction analysis and normalized to 18S rRNA. (B) Transient transfection of a DARS-AS1 promoter [wildtype (WT) or mutant] luciferase transcriptional reporter and plasmid overexpressing HIF-1α/control vector. The luciferase values were determined from cell lysates by normalization to renilla luciferase. The pGL3 control vector containing the HRE promoter was used as a positive control. The data represent the average and standard deviation of three independent experiments. (C) Chromatin immunoprecipitation assay certified the binding of HIF-1 with the predicted two HIF response element (HRE) regions; LDHA was used as a positive control. (D) RNA immunoprecipitation experiments were performed using a Flag-antibody (exogenous HIF-1α protein with Flag-tag), and specific primers to detect DARS-AS1. (E) The expression of HIF-1α in the RPMI 8226 cells overexpressing DARS-AS1 in a hypoxic environment was analyzed by immunoblotting. (F) DARS-AS1-knockdown RPMI 8226 cells were treated with dimethylsulfoxide (DMSO, 1:1000) or MG132 (5 μM) for 6 h. The expression of HIF-1α under hypoxic conditions was analyzed by immunoblotting. (G) Proposed mode of action of DARS-AS1 in modulating myeloma tumorigenesis in a hypoxic environment. DARS-AS1 is directly regulated by HIF-1 and, in turn, promotes the expression of HIF-1α in myeloma. RBM39 mediates DARS-AS1-induced activation of mammalian target of rapamycin (mTOR) signaling. DARS-AS1 stabilizes the RBM39 by interfering with E3 ligase RNF147-mediated ubiquitination. HIF-1/DARS-AS1/RBM39 signaling pathways are involved in the tumorigenesis of myeloma.
Figure Legend Snippet: HIF-1 directly upregulates the expression of DARS-AS1, which may, in turn, enhance the expression of HIF-1α. (A) HIF-1α or HIF-2α was knocked down or overexpressed in 293T cells. DARS-AS1 expression levels were determined by quantitative polymerase chain reaction analysis and normalized to 18S rRNA. (B) Transient transfection of a DARS-AS1 promoter [wildtype (WT) or mutant] luciferase transcriptional reporter and plasmid overexpressing HIF-1α/control vector. The luciferase values were determined from cell lysates by normalization to renilla luciferase. The pGL3 control vector containing the HRE promoter was used as a positive control. The data represent the average and standard deviation of three independent experiments. (C) Chromatin immunoprecipitation assay certified the binding of HIF-1 with the predicted two HIF response element (HRE) regions; LDHA was used as a positive control. (D) RNA immunoprecipitation experiments were performed using a Flag-antibody (exogenous HIF-1α protein with Flag-tag), and specific primers to detect DARS-AS1. (E) The expression of HIF-1α in the RPMI 8226 cells overexpressing DARS-AS1 in a hypoxic environment was analyzed by immunoblotting. (F) DARS-AS1-knockdown RPMI 8226 cells were treated with dimethylsulfoxide (DMSO, 1:1000) or MG132 (5 μM) for 6 h. The expression of HIF-1α under hypoxic conditions was analyzed by immunoblotting. (G) Proposed mode of action of DARS-AS1 in modulating myeloma tumorigenesis in a hypoxic environment. DARS-AS1 is directly regulated by HIF-1 and, in turn, promotes the expression of HIF-1α in myeloma. RBM39 mediates DARS-AS1-induced activation of mammalian target of rapamycin (mTOR) signaling. DARS-AS1 stabilizes the RBM39 by interfering with E3 ligase RNF147-mediated ubiquitination. HIF-1/DARS-AS1/RBM39 signaling pathways are involved in the tumorigenesis of myeloma.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Luciferase, Plasmid Preparation, Positive Control, Standard Deviation, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, FLAG-tag, Activation Assay

DARS-AS1 is upregulated in myeloma cells in a hypoxic environment. (A) RNA-sequencing analysis revealed 77 differentially expressed long non-coding (lnc)RNA between myeloma cells cultured in normoxic or hypoxic conditions. (B) Twenty-two upregulated lncRNA in U266 cells were successfully identified by quantitative polymerase chain reaction (Q-PCR). (C) Multiple myeloma cell lines were cultured under hypoxic culture conditions (1% oxygen concentration) for 48 h and DARS-AS1 expression levels were determined by Q-PCR and normalized to those of 18S rRNA. (D) CD38 + cells derived from bone marrow of patients with active myeloma and normal human bone marrow mononuclear cells were cultured in a hypoxic environment (1% oxygen concentration) for 24 h. The DARS-AS1 expression levels were determined by Q-PCR and normalized to those of 18S rRNA. BMMC-HD: bone marrow mononuclear cells from healthy donors.
Figure Legend Snippet: DARS-AS1 is upregulated in myeloma cells in a hypoxic environment. (A) RNA-sequencing analysis revealed 77 differentially expressed long non-coding (lnc)RNA between myeloma cells cultured in normoxic or hypoxic conditions. (B) Twenty-two upregulated lncRNA in U266 cells were successfully identified by quantitative polymerase chain reaction (Q-PCR). (C) Multiple myeloma cell lines were cultured under hypoxic culture conditions (1% oxygen concentration) for 48 h and DARS-AS1 expression levels were determined by Q-PCR and normalized to those of 18S rRNA. (D) CD38 + cells derived from bone marrow of patients with active myeloma and normal human bone marrow mononuclear cells were cultured in a hypoxic environment (1% oxygen concentration) for 24 h. The DARS-AS1 expression levels were determined by Q-PCR and normalized to those of 18S rRNA. BMMC-HD: bone marrow mononuclear cells from healthy donors.

Techniques Used: RNA Sequencing Assay, Cell Culture, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Concentration Assay, Expressing, Derivative Assay

DARS-AS1 interacts with RBM39. (A) Schematic of the RNA DARS-AS1 pulldown experiment. (B) RNA electrophoresis showed the sense and antisense DARS-AS1 in vitro .(C) Western blotting analysis of the proteins from the proteomics screen after pulldown. (D) RNA immunoprecipitation experiments were performed using a Flag antibody (exogenous RBM39 protein with Flag-tag), and specific primers to detect DARS-AS1 or β-actin.
Figure Legend Snippet: DARS-AS1 interacts with RBM39. (A) Schematic of the RNA DARS-AS1 pulldown experiment. (B) RNA electrophoresis showed the sense and antisense DARS-AS1 in vitro .(C) Western blotting analysis of the proteins from the proteomics screen after pulldown. (D) RNA immunoprecipitation experiments were performed using a Flag antibody (exogenous RBM39 protein with Flag-tag), and specific primers to detect DARS-AS1 or β-actin.

Techniques Used: Electrophoresis, In Vitro, Western Blot, Immunoprecipitation, FLAG-tag

25) Product Images from "Long intergenic non-coding RNA APOC1P1-3 inhibits apoptosis by decreasing α-tubulin acetylation in breast cancer"

Article Title: Long intergenic non-coding RNA APOC1P1-3 inhibits apoptosis by decreasing α-tubulin acetylation in breast cancer

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.142

APOC1P1-3 can bind to tubulin and modify its acetylation levels. ( a ) RNA pull-down to detect the specific combining proteins of APOC1P1-3 , silver stain of the SDS-PAGE gel showed that there was a specific bond between 34 and 55 kDa. The bond was cut to mass spectrometry analysis and was identified as tubulin. ( b ) Western blot to validate the mass spectrometry results in MDA-MB-231 and MCF7 cell lines. ( c ) Relative RIP experiments were performed with anti-tubulin antibodies on extracts from MDA-MB-231 and MCF7 cells, respectively, with IgG as a negative control. The purified RNA was used for qPCR analysis, and the enrichment of APOC1P1-3 was normalized to input. * P
Figure Legend Snippet: APOC1P1-3 can bind to tubulin and modify its acetylation levels. ( a ) RNA pull-down to detect the specific combining proteins of APOC1P1-3 , silver stain of the SDS-PAGE gel showed that there was a specific bond between 34 and 55 kDa. The bond was cut to mass spectrometry analysis and was identified as tubulin. ( b ) Western blot to validate the mass spectrometry results in MDA-MB-231 and MCF7 cell lines. ( c ) Relative RIP experiments were performed with anti-tubulin antibodies on extracts from MDA-MB-231 and MCF7 cells, respectively, with IgG as a negative control. The purified RNA was used for qPCR analysis, and the enrichment of APOC1P1-3 was normalized to input. * P

Techniques Used: Silver Staining, SDS Page, Mass Spectrometry, Western Blot, Multiple Displacement Amplification, Negative Control, Purification, Real-time Polymerase Chain Reaction

26) Product Images from "Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2"

Article Title: Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2

Journal: Protein & Cell

doi: 10.1007/s13238-017-0448-9

Characterization of a ribonuclease activity in the mitochondrial IMS . (A) Immunoblots of total mitochondria and fractions. Mitochondria were separated into four fractions: total soluble (including IMS and Matrix), total membrane, IMS and matrix. Immunoblotting was performed using antibodies for Mortalin (Matrix), TIM23 (Inner membrane), DDP2 (IMS), and TOM40 (Outer membrane). (B) Four mitochondrial fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. (C) No ribonuclease activity localizes at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in mitoprep buffer (M buffer) or hypotonic buffer (H buffer) that ruptures the mitochondrial outer membrane. Biotinylated UCP2 RNA was added to the mixture and incubated at 37°C for 1 min or 5 min before the reaction was terminated. (D) IMS ribonuclease activity was tested for its sensitivity to EDTA (2 mmol/L) and Cu 2+ (0.5 mmol/L) using biotinylated UCP2 mRNA as a substrate. (E) IMS ribonuclease activity was tested for its sensitivity to different concentrations of Cu 2+ (0.5 mmol/L and 5 mmol/L), and Mg 2+ (0.5 mmol/L and 10 mmol/L) using RNAs purified from isolated mitochondria as substrates. (F) IMS ribonuclease activity and RNaseI were tested for sensitivity to different concentrations of ATP (0 mmol/L, 5 mmol/L, and 10 mmol/L). (G) IMS ribonuclease activity was tested for sensitivity to Proteinase K (ProK), EDTA (2 mmol/L), Cu 2+ (0.5 mmol/L), and heat (90°C 10 min)
Figure Legend Snippet: Characterization of a ribonuclease activity in the mitochondrial IMS . (A) Immunoblots of total mitochondria and fractions. Mitochondria were separated into four fractions: total soluble (including IMS and Matrix), total membrane, IMS and matrix. Immunoblotting was performed using antibodies for Mortalin (Matrix), TIM23 (Inner membrane), DDP2 (IMS), and TOM40 (Outer membrane). (B) Four mitochondrial fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. (C) No ribonuclease activity localizes at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in mitoprep buffer (M buffer) or hypotonic buffer (H buffer) that ruptures the mitochondrial outer membrane. Biotinylated UCP2 RNA was added to the mixture and incubated at 37°C for 1 min or 5 min before the reaction was terminated. (D) IMS ribonuclease activity was tested for its sensitivity to EDTA (2 mmol/L) and Cu 2+ (0.5 mmol/L) using biotinylated UCP2 mRNA as a substrate. (E) IMS ribonuclease activity was tested for its sensitivity to different concentrations of Cu 2+ (0.5 mmol/L and 5 mmol/L), and Mg 2+ (0.5 mmol/L and 10 mmol/L) using RNAs purified from isolated mitochondria as substrates. (F) IMS ribonuclease activity and RNaseI were tested for sensitivity to different concentrations of ATP (0 mmol/L, 5 mmol/L, and 10 mmol/L). (G) IMS ribonuclease activity was tested for sensitivity to Proteinase K (ProK), EDTA (2 mmol/L), Cu 2+ (0.5 mmol/L), and heat (90°C 10 min)

Techniques Used: Activity Assay, Western Blot, Isolation, Incubation, Purification

Characterization of mtRNA degradation using an in organello system . (A) Coomassie staining of human PNPASE samples purified from HEK293 mitochondria (HEK mito) or E . coli . For PNPASE purified from HEK mitochondria, two concentrations of samples were loaded. Con: same volume of eluate from cells harboring the empty vector; PNP: eluate from PNPASE-HisPC expressing cells. (B) PNPASE purified from E . coli or HEK mitochondria was incubated with Biotin-labeled UCP2 RNA. The lower panel shows immunoblotting of PNPASE. (C) PNPASE purified from E . coli or HEK mitochondria was incubated with total cytosolic RNA and the samples were resolved on an agarose gel. The lower panel shows immunoblotting of PNPASE. (D) Mitochondria were separated into total soluble and total membrane and the two fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. Lower panels show Immunoblotting of PNPASE (Membrane) and DDP2 (Soluble). (E) Effect of temperature on in organello mtRNA degradation. Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. The three numbers (1, 2, and 3) represent three time points (5 min, 25 min, and 45 min). Top panel on the left shows the remaining labeled mtRNAs. Bottom panel is an immunoblot of mitochondrial protein Mortalin showing the amount of mitochondria taken out at each time point. Right panel shows the quantification of labeled mtRNAs ( n = 3). (F) Effect of pH on in organello mtRNA degradation. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified) or pH 6.5. (G) Effect of Cu 2+ on in organello mtRNA degradation. Two concentrations (0 mmol/L and 0.5 mmol/L) of Cu 2+ were used. (H) Effect of Mg 2+ on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 20 mmol/L) of Mg 2+ were used. (I) Effect of ATP on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 8 mmol/L) of ATP were used. Statistical comparisons are performed using unpaired t -tests ( n = 3 if not specified); * P
Figure Legend Snippet: Characterization of mtRNA degradation using an in organello system . (A) Coomassie staining of human PNPASE samples purified from HEK293 mitochondria (HEK mito) or E . coli . For PNPASE purified from HEK mitochondria, two concentrations of samples were loaded. Con: same volume of eluate from cells harboring the empty vector; PNP: eluate from PNPASE-HisPC expressing cells. (B) PNPASE purified from E . coli or HEK mitochondria was incubated with Biotin-labeled UCP2 RNA. The lower panel shows immunoblotting of PNPASE. (C) PNPASE purified from E . coli or HEK mitochondria was incubated with total cytosolic RNA and the samples were resolved on an agarose gel. The lower panel shows immunoblotting of PNPASE. (D) Mitochondria were separated into total soluble and total membrane and the two fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. Lower panels show Immunoblotting of PNPASE (Membrane) and DDP2 (Soluble). (E) Effect of temperature on in organello mtRNA degradation. Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. The three numbers (1, 2, and 3) represent three time points (5 min, 25 min, and 45 min). Top panel on the left shows the remaining labeled mtRNAs. Bottom panel is an immunoblot of mitochondrial protein Mortalin showing the amount of mitochondria taken out at each time point. Right panel shows the quantification of labeled mtRNAs ( n = 3). (F) Effect of pH on in organello mtRNA degradation. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified) or pH 6.5. (G) Effect of Cu 2+ on in organello mtRNA degradation. Two concentrations (0 mmol/L and 0.5 mmol/L) of Cu 2+ were used. (H) Effect of Mg 2+ on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 20 mmol/L) of Mg 2+ were used. (I) Effect of ATP on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 8 mmol/L) of ATP were used. Statistical comparisons are performed using unpaired t -tests ( n = 3 if not specified); * P

Techniques Used: Staining, Purification, Plasmid Preparation, Expressing, Incubation, Labeling, Agarose Gel Electrophoresis, Activity Assay

Characterization of RNASET2 purified from HEK mitochondria . (A) Dual-tag purification of RNASET2 (His and HA). Purification was performed using IMS from control HEK cells (C) or RNASET2-overexpressing cells (T2) under native condition. (B) Ribonuclease activity were examined in IMS samples and the purification samples (Eluate) from control HEK cells (C) or RNASET2-overexpressing cells (T2) using biotinylated UCP2 mRNA as a substrate. The sensitivity of these activities to Cu 2+ (0.5 mmol/L) and proteinase K (ProK) was also tested. (C) RNASET2 was purified from RNASET2-overexpressing mitochondria under denaturing condition and checked for ribonuclease activity using RNA purified from isolated mitochondria as substrates; C (control pulldown from HEK mitochondria) and T2 (RNASET2). (D) RNASET2s purified under native conditions and denaturing conditions had the same responses to proteinase K (ProK) and Cu 2+ treatment. Lower panel is a coomassie staining gel of mitochondrial lysates as a positive control for proteinase K treatment. (E) Effect of temperature on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA (50 ng). Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. RNAs purified from isolated mitochondria were used as substrates. Con (control pulldown from HEK mitochondria). (F) Effect of pH on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified), pH 6.5 or pH 5.5. (G) Effect of ATP on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA. (H) Effect of Mg 2+ and Cu 2+ on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA
Figure Legend Snippet: Characterization of RNASET2 purified from HEK mitochondria . (A) Dual-tag purification of RNASET2 (His and HA). Purification was performed using IMS from control HEK cells (C) or RNASET2-overexpressing cells (T2) under native condition. (B) Ribonuclease activity were examined in IMS samples and the purification samples (Eluate) from control HEK cells (C) or RNASET2-overexpressing cells (T2) using biotinylated UCP2 mRNA as a substrate. The sensitivity of these activities to Cu 2+ (0.5 mmol/L) and proteinase K (ProK) was also tested. (C) RNASET2 was purified from RNASET2-overexpressing mitochondria under denaturing condition and checked for ribonuclease activity using RNA purified from isolated mitochondria as substrates; C (control pulldown from HEK mitochondria) and T2 (RNASET2). (D) RNASET2s purified under native conditions and denaturing conditions had the same responses to proteinase K (ProK) and Cu 2+ treatment. Lower panel is a coomassie staining gel of mitochondrial lysates as a positive control for proteinase K treatment. (E) Effect of temperature on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA (50 ng). Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. RNAs purified from isolated mitochondria were used as substrates. Con (control pulldown from HEK mitochondria). (F) Effect of pH on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified), pH 6.5 or pH 5.5. (G) Effect of ATP on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA. (H) Effect of Mg 2+ and Cu 2+ on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA

Techniques Used: Purification, Activity Assay, Isolation, Staining, Positive Control

27) Product Images from "A novel positive feedback regulation between long noncoding RNA UICC and IL-6/STAT3 signaling promotes cervical cancer progression"

Article Title: A novel positive feedback regulation between long noncoding RNA UICC and IL-6/STAT3 signaling promotes cervical cancer progression

Journal: American Journal of Cancer Research

doi:

lnc-UICC interacts with p-STAT3 protein. A. The RIP assay was performed to determine the interaction between lnc-UICC and p-STAT3. The IgG was taken as negative control. B. The RNA pull down assay was performed to determine the interaction between lnc-UICC and p-STAT3. Antisense (AS) lnc-UICC was was taken as negative control. C. HeLa cells with or without lnc-UICC overexpression were treated with CHX (100 mg/ml) for the indicated time points. The cell lysates were examined by immunoblotting (up panel). A plot of normalized amount of p-STAT3 protein is shown (down panel). D. HeLa cells with or without lnc-UICC knockdown were treated with CHX (100 mg/ml) for the indicated time points. The cell lysates were examined by immunoblotting (up panel). A plot of normalized amount of p-STAT3 protein is shown (down panel). E. Western blot of p-STAT3-associated ubiquitination in cervical cancer cells with lnc-UICC overexpression (left panel) or knockdown (right panel). F. Western blot of p-STAT3 expression in SiHa-Con and SiHa-KD1 cells treated with vehicle control or MG132. *P
Figure Legend Snippet: lnc-UICC interacts with p-STAT3 protein. A. The RIP assay was performed to determine the interaction between lnc-UICC and p-STAT3. The IgG was taken as negative control. B. The RNA pull down assay was performed to determine the interaction between lnc-UICC and p-STAT3. Antisense (AS) lnc-UICC was was taken as negative control. C. HeLa cells with or without lnc-UICC overexpression were treated with CHX (100 mg/ml) for the indicated time points. The cell lysates were examined by immunoblotting (up panel). A plot of normalized amount of p-STAT3 protein is shown (down panel). D. HeLa cells with or without lnc-UICC knockdown were treated with CHX (100 mg/ml) for the indicated time points. The cell lysates were examined by immunoblotting (up panel). A plot of normalized amount of p-STAT3 protein is shown (down panel). E. Western blot of p-STAT3-associated ubiquitination in cervical cancer cells with lnc-UICC overexpression (left panel) or knockdown (right panel). F. Western blot of p-STAT3 expression in SiHa-Con and SiHa-KD1 cells treated with vehicle control or MG132. *P

Techniques Used: Negative Control, Pull Down Assay, Over Expression, Western Blot, Expressing

lnc-UICC increases IL-6 transcription. (A and B) mRNA level of IL-6 in lnc-UICC overexpressed (A) or knockdown (B) xenograft. (C and D) Media from the indicated cultured lnc-UICC overexpressed (C) or lnc-UICC knockdown (D) cervical cancer cells was collected and subjected to IL-6 ELISA assays. (E and F) mRNA level of IL-6 in lnc-UICC overexpressed (C) or knockdown (D) cervical cancer cells. (G) The sequence of the predicted lnc-UICC binding locus in IL-6 promoter is shown. The sequences of oligoribonucleotides (RNA oligo) and oligodeoxyonucleotides (DNA decoy) are shown. (H) qRT-PCR analysis of lnc-UICC pulled down by biotin-labeled antisense oligodeoxynucleotide (Biotin-AS) or sense oligodeoxynucleotide (Biotin-S) control in HeLa cells. (I) Transcript of IL-6 was measured in HeLa-Con and HeLa-OE cells transfected with 200 nM oligodeoxynucleotides negative control (ODN NC), lnc-lnc-UICC oligodeoxynucleotides decoy (UICC decoy) or mutated oligodeoxynucleotides (ODN mut) was measured by qRT-PCR analysis. (J) STAT3 activity was measured in HeLa-Con and HeLa-OE cells transfected with 200 nM oligodeoxynucleotides negative control (ODN NC), lnc-lnc-UICC oligodeoxynucleotides decoy (UICC decoy) or mutated oligodeoxynucleotides (ODN mut) by luciferase activity analysis. (K) Transcript of IL-6 in SiHa-Con and SiHa-KD1 cells transfected with oligoribonucleotide negative control (oligo NC), oligo mimics or oligo mut (200 nM) by qRT-PCR analysis. (L) STAT3 activity in SiHa-Con and SiHa-KD1 cells transfected with oligoribonucleotide negative control (oligo NC), oligo mimics or oligo mut (200 nM) was measured by luciferase activity analysis. *P
Figure Legend Snippet: lnc-UICC increases IL-6 transcription. (A and B) mRNA level of IL-6 in lnc-UICC overexpressed (A) or knockdown (B) xenograft. (C and D) Media from the indicated cultured lnc-UICC overexpressed (C) or lnc-UICC knockdown (D) cervical cancer cells was collected and subjected to IL-6 ELISA assays. (E and F) mRNA level of IL-6 in lnc-UICC overexpressed (C) or knockdown (D) cervical cancer cells. (G) The sequence of the predicted lnc-UICC binding locus in IL-6 promoter is shown. The sequences of oligoribonucleotides (RNA oligo) and oligodeoxyonucleotides (DNA decoy) are shown. (H) qRT-PCR analysis of lnc-UICC pulled down by biotin-labeled antisense oligodeoxynucleotide (Biotin-AS) or sense oligodeoxynucleotide (Biotin-S) control in HeLa cells. (I) Transcript of IL-6 was measured in HeLa-Con and HeLa-OE cells transfected with 200 nM oligodeoxynucleotides negative control (ODN NC), lnc-lnc-UICC oligodeoxynucleotides decoy (UICC decoy) or mutated oligodeoxynucleotides (ODN mut) was measured by qRT-PCR analysis. (J) STAT3 activity was measured in HeLa-Con and HeLa-OE cells transfected with 200 nM oligodeoxynucleotides negative control (ODN NC), lnc-lnc-UICC oligodeoxynucleotides decoy (UICC decoy) or mutated oligodeoxynucleotides (ODN mut) by luciferase activity analysis. (K) Transcript of IL-6 in SiHa-Con and SiHa-KD1 cells transfected with oligoribonucleotide negative control (oligo NC), oligo mimics or oligo mut (200 nM) by qRT-PCR analysis. (L) STAT3 activity in SiHa-Con and SiHa-KD1 cells transfected with oligoribonucleotide negative control (oligo NC), oligo mimics or oligo mut (200 nM) was measured by luciferase activity analysis. *P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Sequencing, Binding Assay, Quantitative RT-PCR, Labeling, Transfection, Negative Control, Activity Assay, Luciferase

IL-6/STAT3 activates lnc-UICC transcription to form a positive feed-back regulatory loop. A. The cervical cancer cells were treated with 10 ng/mL rhIL-6 for 12 hours. The lnc-UICC was then detected by qRT-PCR. B. The cervical cancer cells were transfected with IL-6 siRNA. After 48h, the lnc-UICC was then detected by qRT-PCR. C. Schematic diagram of canonical STAT3-binding motif (JASPAR Database) and three potential STAT3 responsive elements (E1, E2, and E3) in the lnc-UICC promoter region. D. lnc-UICC RNA levels when 20 μM STAT3 inhibitor and 10 ng/mL rhIL-6 was used to treat cells. E. lnc-UICC RNA levels when STAT3 siRNA and 10 ng/mL rhIL-6 was used to treat cells. F. lnc-UICC promoter luciferase activity when 20 μM STAT3 inhibitor and 10 ng/mL rhIL-6 was used to treat cells. G. lnc-UICC promoter luciferase activity and 10 ng/mL rhIL-6 was used to treat cells. H. Three predicted STAT3-binding sites of the lnc-UICC promoter was individually deleted and named E1-Del, E2-Del, and E3-Del. Luciferase assay was employed to detect transcriptional activities of the three lnc-UICC promoter deletion mutants when STAT3 expression was enforced in HeLa cells. FL indicates Full-length lnc-UICC promoter sequence. I. ChIP assays showed that STAT3 bound to the E2 element of lnc-UICC promoter. IgG served as a negative control. *P
Figure Legend Snippet: IL-6/STAT3 activates lnc-UICC transcription to form a positive feed-back regulatory loop. A. The cervical cancer cells were treated with 10 ng/mL rhIL-6 for 12 hours. The lnc-UICC was then detected by qRT-PCR. B. The cervical cancer cells were transfected with IL-6 siRNA. After 48h, the lnc-UICC was then detected by qRT-PCR. C. Schematic diagram of canonical STAT3-binding motif (JASPAR Database) and three potential STAT3 responsive elements (E1, E2, and E3) in the lnc-UICC promoter region. D. lnc-UICC RNA levels when 20 μM STAT3 inhibitor and 10 ng/mL rhIL-6 was used to treat cells. E. lnc-UICC RNA levels when STAT3 siRNA and 10 ng/mL rhIL-6 was used to treat cells. F. lnc-UICC promoter luciferase activity when 20 μM STAT3 inhibitor and 10 ng/mL rhIL-6 was used to treat cells. G. lnc-UICC promoter luciferase activity and 10 ng/mL rhIL-6 was used to treat cells. H. Three predicted STAT3-binding sites of the lnc-UICC promoter was individually deleted and named E1-Del, E2-Del, and E3-Del. Luciferase assay was employed to detect transcriptional activities of the three lnc-UICC promoter deletion mutants when STAT3 expression was enforced in HeLa cells. FL indicates Full-length lnc-UICC promoter sequence. I. ChIP assays showed that STAT3 bound to the E2 element of lnc-UICC promoter. IgG served as a negative control. *P

Techniques Used: Quantitative RT-PCR, Transfection, Binding Assay, Luciferase, Activity Assay, Expressing, Sequencing, Chromatin Immunoprecipitation, Negative Control

28) Product Images from "Incorporation of the Noncoding roX RNAs Alters the Chromatin-Binding Specificity of the Drosophila MSL1/MSL2 Complex ▿ MSL1/MSL2 Complex ▿ †"

Article Title: Incorporation of the Noncoding roX RNAs Alters the Chromatin-Binding Specificity of the Drosophila MSL1/MSL2 Complex ▿ MSL1/MSL2 Complex ▿ †

Journal:

doi: 10.1128/MCB.00910-07

Association of roX1 RNA with polytene chromosomes in hsp70 - msl2 lines. (A to H) Single-stranded biotinylated roX1 probe was hybridized to salivary gland nuclei from female larvae that express the indicated version of MSL2 and were detected with fluorescein-avidin
Figure Legend Snippet: Association of roX1 RNA with polytene chromosomes in hsp70 - msl2 lines. (A to H) Single-stranded biotinylated roX1 probe was hybridized to salivary gland nuclei from female larvae that express the indicated version of MSL2 and were detected with fluorescein-avidin

Techniques Used: Avidin-Biotin Assay

roX RNA levels in hsp - msl2 lines. (A) Salivary gland nuclei from female larvae that express MSL2(1-193)F were stained with anti-MSL2 (top) and counterstained with DAPI (lower). MSL2(1-193)F bound to 3F and 10C, the locations of the roX1 and roX2 genes,
Figure Legend Snippet: roX RNA levels in hsp - msl2 lines. (A) Salivary gland nuclei from female larvae that express MSL2(1-193)F were stained with anti-MSL2 (top) and counterstained with DAPI (lower). MSL2(1-193)F bound to 3F and 10C, the locations of the roX1 and roX2 genes,

Techniques Used: Staining

29) Product Images from "piRNA-mediated transgenerational inheritance of an acquired trait"

Article Title: piRNA-mediated transgenerational inheritance of an acquired trait

Journal: Genome Research

doi: 10.1101/gr.136614.111

The rescue of SF sterility induced by aging treatment of the maternal ancestors correlates with I-element repression. ( A ) Schematic representation of the biological model. The reactive (R) Cha strain was divided into two stocks and bred during at least five successive short or long generations to produce isogenic R L and R H stocks, respectively. Short and long open arrows correspond to the age of the egg-laying females (3-d-old and 25- to 30-d-old, respectively) used to produce the subsequent generation. Three-day-old females from both stocks were crossed with Cha-I inducer males to generate SF L and SF H females. The blue asterisks highlight flies the ovaries and eggs of which were used to perform small RNA sequencing. (3d) 3-d-old females; (25-30d) 25- to 30-d-old females. ( B ) Histogram showing the hatching percentage of eggs from SF L 3d (white) and SF H 3d (gray) females (bars, mean ± SD; Wilcoxon signed-rank test P = 10 −12 , n = 33 vials). ( C ) RNA FISH on whole-mount preparations of ovaries from SF L 3d ( left panel) and SF H 3d flies ( right panel) using a FITC-conjugated antibody against a biotin-labeled antisense riboprobe specific for I-element sense transcripts. I-element sense transcripts were detected (green) only in oocytes of SF L ovaries. Ovaries were counterstained with DAPI (blue).
Figure Legend Snippet: The rescue of SF sterility induced by aging treatment of the maternal ancestors correlates with I-element repression. ( A ) Schematic representation of the biological model. The reactive (R) Cha strain was divided into two stocks and bred during at least five successive short or long generations to produce isogenic R L and R H stocks, respectively. Short and long open arrows correspond to the age of the egg-laying females (3-d-old and 25- to 30-d-old, respectively) used to produce the subsequent generation. Three-day-old females from both stocks were crossed with Cha-I inducer males to generate SF L and SF H females. The blue asterisks highlight flies the ovaries and eggs of which were used to perform small RNA sequencing. (3d) 3-d-old females; (25-30d) 25- to 30-d-old females. ( B ) Histogram showing the hatching percentage of eggs from SF L 3d (white) and SF H 3d (gray) females (bars, mean ± SD; Wilcoxon signed-rank test P = 10 −12 , n = 33 vials). ( C ) RNA FISH on whole-mount preparations of ovaries from SF L 3d ( left panel) and SF H 3d flies ( right panel) using a FITC-conjugated antibody against a biotin-labeled antisense riboprobe specific for I-element sense transcripts. I-element sense transcripts were detected (green) only in oocytes of SF L ovaries. Ovaries were counterstained with DAPI (blue).

Techniques Used: Sterility, RNA Sequencing Assay, Fluorescence In Situ Hybridization, Labeling

The amount of maternally deposited I-element piRNAs in eggs determines the amount of piRNAs in female ovaries. The left and right panels schematize the biogenesis of I-element piRNAs in the R L and R H stocks, respectively. Secondary antisense piRNAs (green arrows with uridine in the first position [1U]) are abundant in the eggs laid by aged ancestors, leading to frequent ping-pong loop initiation in ovarian germ cells (thicker blue arrow in the right panel) and, therefore, abundant ovarian secondary piRNA production and maternal piRNA deposition in the next R H generations (gray dashed line). The amount of the RNA precursors (black lines) that contain defective I-element fragments (orange arrows) is similar in both R L and R H stocks. The amount of secondary piRNAs accumulated in the ovary is therefore not limited by the amount of RNA precursors but by the amount of the maternally deposited piRNAs. Since the maternal deposit only partially reflects the secondary ovarian piRNA accumulation, the system progressively tends to the stable equilibrium of the R L stock if the environmental changes are stopped.
Figure Legend Snippet: The amount of maternally deposited I-element piRNAs in eggs determines the amount of piRNAs in female ovaries. The left and right panels schematize the biogenesis of I-element piRNAs in the R L and R H stocks, respectively. Secondary antisense piRNAs (green arrows with uridine in the first position [1U]) are abundant in the eggs laid by aged ancestors, leading to frequent ping-pong loop initiation in ovarian germ cells (thicker blue arrow in the right panel) and, therefore, abundant ovarian secondary piRNA production and maternal piRNA deposition in the next R H generations (gray dashed line). The amount of the RNA precursors (black lines) that contain defective I-element fragments (orange arrows) is similar in both R L and R H stocks. The amount of secondary piRNAs accumulated in the ovary is therefore not limited by the amount of RNA precursors but by the amount of the maternally deposited piRNAs. Since the maternal deposit only partially reflects the secondary ovarian piRNA accumulation, the system progressively tends to the stable equilibrium of the R L stock if the environmental changes are stopped.

Techniques Used:

30) Product Images from "Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins"

Article Title: Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins

Journal: Genome Biology

doi: 10.1186/gb-2011-12-1-r3

Gis2p preferentially binds to coding sequences that bear GAN repeats . (a) Conserved sequence element in the ORF of Gis2p targets identified with MEME. The E-value reflects the probability to detect the motif by chance. (b) RNA-protein complexes formed between biotinylated RNA fragments and Gis2-TAP were purified on streptavidin beads and monitored by immunoblot analysis. Representative experiments from at least three biological replicates are shown. Biotin labeled fragments comprising the 5'-UTRs (lanes 2 and 5), ORFs (lanes 3 and 6), and 3'-UTRs (lanes 4 and 7) of Erv25 and Fcy1 were incubated with extracts of Gis2-TAP expressing cells (lane 1). Eno2-5'UTR (lane 8) is a negative control RNA derived from the 'non-target' ENO2 (lane 8) and a sample without RNA (lane 9) was used to control for RNA-independent binding to the beads. (c) RNA pull-downs with RNA fragments derived from NOP53 . Nop53-GAN (lanes 3 and 7 to 9) contains a GAN-rich sequence element whereas the similarly sized fragment Nop53-ctrl does not (lanes 4 and 10). Erv25-ORF (lane 2) and Eno2-ORF (lane 5) are positive and negative control RNAs, respectively. Binding of Gis2-TAP to Nop53-GAN was competed with a ten-fold excess of non-biotinylated Nop53-GAN (lane 8) but not with excess of Nop53-ctrl (lane 9). (d) RNA pull-downs with two fragments derived from the RAS2 ORF. Biotinylated RNAs were incubated with extracts from yeast cells expressing Gis2-TAP (eGis2-TAP, lanes 1 to 3) or with Gis2-His expressed and purified from Escherichia coli (pGis2-His, lanes 4 and 5). A fragment derived from the ORF of SNF5 was used as a negative control RNA (lane 3).
Figure Legend Snippet: Gis2p preferentially binds to coding sequences that bear GAN repeats . (a) Conserved sequence element in the ORF of Gis2p targets identified with MEME. The E-value reflects the probability to detect the motif by chance. (b) RNA-protein complexes formed between biotinylated RNA fragments and Gis2-TAP were purified on streptavidin beads and monitored by immunoblot analysis. Representative experiments from at least three biological replicates are shown. Biotin labeled fragments comprising the 5'-UTRs (lanes 2 and 5), ORFs (lanes 3 and 6), and 3'-UTRs (lanes 4 and 7) of Erv25 and Fcy1 were incubated with extracts of Gis2-TAP expressing cells (lane 1). Eno2-5'UTR (lane 8) is a negative control RNA derived from the 'non-target' ENO2 (lane 8) and a sample without RNA (lane 9) was used to control for RNA-independent binding to the beads. (c) RNA pull-downs with RNA fragments derived from NOP53 . Nop53-GAN (lanes 3 and 7 to 9) contains a GAN-rich sequence element whereas the similarly sized fragment Nop53-ctrl does not (lanes 4 and 10). Erv25-ORF (lane 2) and Eno2-ORF (lane 5) are positive and negative control RNAs, respectively. Binding of Gis2-TAP to Nop53-GAN was competed with a ten-fold excess of non-biotinylated Nop53-GAN (lane 8) but not with excess of Nop53-ctrl (lane 9). (d) RNA pull-downs with two fragments derived from the RAS2 ORF. Biotinylated RNAs were incubated with extracts from yeast cells expressing Gis2-TAP (eGis2-TAP, lanes 1 to 3) or with Gis2-His expressed and purified from Escherichia coli (pGis2-His, lanes 4 and 5). A fragment derived from the ORF of SNF5 was used as a negative control RNA (lane 3).

Techniques Used: Sequencing, Purification, Labeling, Incubation, Expressing, Negative Control, Derivative Assay, Binding Assay

Gis2p and ZNF9 bind specifically to GWW repeats in RNA and to G-rich sequences in ssDNA . RNA-protein complexes formed between biotinylated RNAs and yeast extracts expressing Gis2-TAP or ZNF9-TAP (eGis2/eZNF9) or recombinant Gis2-His or ZNF9-His purified from E. coli (pGis2/pZNF9) were captured with streptavidin beads and visualized by immunoblot analysis with specific antibodies detecting the TAP or His tag. Representative experiments from at least three biological replicates are shown. (a) RNA pull-downs with short biotinylated RNAs bearing different nucleotide triplet repeats (lanes 2 to 7). The consensus sequence for protein-RNA interaction is depicted on the right. (b) Testing different sizes of GWW loops for interaction with Gis2p/ZNF9 (lanes 1 to 6). The predicted stem-loop structure with varying sizes of GWW-loops is shown to the left. (c) RNA pull-downs after the addition of ten-fold excess of non-labeled competitor RNA (lanes 3 to 5) or ssDNA (lanes 6, 7, and 12 to 14). No RNA was added to control for unspecific binding of proteins to the beads (lanes 8 and 11). (d) Binding ZNF9 to human RNAs containing at least three GWW repeats in the coding region (lanes 2 to 4). (GAUGAA) 5 was used as positive control (lane 5), and (GAUGCU) 5 as negative control (lane 7). Binding of ZNF9 to MYH4 RNA was efficiently competed with ten-fold excess of unlabeled (GAUGAA) 5 RNA (lane 6). A reaction without RNA is shown in lane 8.
Figure Legend Snippet: Gis2p and ZNF9 bind specifically to GWW repeats in RNA and to G-rich sequences in ssDNA . RNA-protein complexes formed between biotinylated RNAs and yeast extracts expressing Gis2-TAP or ZNF9-TAP (eGis2/eZNF9) or recombinant Gis2-His or ZNF9-His purified from E. coli (pGis2/pZNF9) were captured with streptavidin beads and visualized by immunoblot analysis with specific antibodies detecting the TAP or His tag. Representative experiments from at least three biological replicates are shown. (a) RNA pull-downs with short biotinylated RNAs bearing different nucleotide triplet repeats (lanes 2 to 7). The consensus sequence for protein-RNA interaction is depicted on the right. (b) Testing different sizes of GWW loops for interaction with Gis2p/ZNF9 (lanes 1 to 6). The predicted stem-loop structure with varying sizes of GWW-loops is shown to the left. (c) RNA pull-downs after the addition of ten-fold excess of non-labeled competitor RNA (lanes 3 to 5) or ssDNA (lanes 6, 7, and 12 to 14). No RNA was added to control for unspecific binding of proteins to the beads (lanes 8 and 11). (d) Binding ZNF9 to human RNAs containing at least three GWW repeats in the coding region (lanes 2 to 4). (GAUGAA) 5 was used as positive control (lane 5), and (GAUGCU) 5 as negative control (lane 7). Binding of ZNF9 to MYH4 RNA was efficiently competed with ten-fold excess of unlabeled (GAUGAA) 5 RNA (lane 6). A reaction without RNA is shown in lane 8.

Techniques Used: Expressing, Recombinant, Purification, Sequencing, Labeling, Binding Assay, Positive Control, Negative Control

31) Product Images from "LncSHRG promotes hepatocellular carcinoma progression by activating HES6"

Article Title: LncSHRG promotes hepatocellular carcinoma progression by activating HES6

Journal: Oncotarget

doi: 10.18632/oncotarget.19906

LncSHRG is essential for SATB1 binding to HES6 promoter (A) SATB1 bound to HES6 promoter (-1100bp∼-900bp) as shown by ChIP assays. χ axis stands for the distance from transcription start site (TSS). (B) SATB1 knockdown inhibited HES6 transcription. Luciferase activity assays were performed with SATB1-silenced sample cells. (C) LncSHRG knockdown impaired SATB1 binding to HES6 promoter (-1100∼-900) as shown by ChIP-qPCR assays. WT or lncSHRG-silenced HCC sample cells were lysed and incubated with anti-SATB1 for ChIP assays. (D) LncSHRG was essential for SATB1-regulated HES6 transcription. Luciferase activity assays were performed with WT or lncSHRG-silenced sample cells. (E) SATB1 overexpression promoted enrichment of RNA pol II on HES6 promoter (-1100∼-900) via an lncSHRG-dependent manner. (F) SATB1 promoted HES6 expression relying on the presence of lncSHRG. WT or lncSHRG-silenced sample cells were transfected with SATB1-overexpressing plasmid or empty plasmid control. Then mRNA levels of HES6 were measured by real time qPCR. *p
Figure Legend Snippet: LncSHRG is essential for SATB1 binding to HES6 promoter (A) SATB1 bound to HES6 promoter (-1100bp∼-900bp) as shown by ChIP assays. χ axis stands for the distance from transcription start site (TSS). (B) SATB1 knockdown inhibited HES6 transcription. Luciferase activity assays were performed with SATB1-silenced sample cells. (C) LncSHRG knockdown impaired SATB1 binding to HES6 promoter (-1100∼-900) as shown by ChIP-qPCR assays. WT or lncSHRG-silenced HCC sample cells were lysed and incubated with anti-SATB1 for ChIP assays. (D) LncSHRG was essential for SATB1-regulated HES6 transcription. Luciferase activity assays were performed with WT or lncSHRG-silenced sample cells. (E) SATB1 overexpression promoted enrichment of RNA pol II on HES6 promoter (-1100∼-900) via an lncSHRG-dependent manner. (F) SATB1 promoted HES6 expression relying on the presence of lncSHRG. WT or lncSHRG-silenced sample cells were transfected with SATB1-overexpressing plasmid or empty plasmid control. Then mRNA levels of HES6 were measured by real time qPCR. *p

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Incubation, Over Expression, Expressing, Transfection, Plasmid Preparation

LncSHRG associates with SATB1 (A) Biotin-RNA pulldowns were performed using biotin-labeled lncSHRG or anti-sense control. Eluted fractions were resolved by SDS-PAGE, followed by silver staining and mass spectrometry. SATB1 was identified as a potential interactive protein of lncSHRG. (B) HCC sample lysates were incubated with anti-SATB1 at 4°C for 4 h, followed by an RNA immunoprecipitation assay. (C) Interaction of His-SATB1 with biotin-lncSHRG was checked by RNA pulldown assays. (D) lncSHRG (nt200∼400) was essential for the interaction with SATB1 as shown by domain mapping and RNA pulldown assays. (E) Biotin-labeled lncSHRG (nt200∼400) probe was incubated with His-SATB1 protein, followed by EMSA assays. (F) LncSHRG (nt200∼400) contributed to cell proliferation by MTT assays. Full-length lncSHRG but not truncation version (deletion of nt200∼400) promotes cell proliferation in HCC samples. (G) LncSHRG (nt200∼400) promoted colony formation. Full-length lncSHRG but not truncation version (deletion of nt200∼400) promotes colony formation of HCC sample cells. *p
Figure Legend Snippet: LncSHRG associates with SATB1 (A) Biotin-RNA pulldowns were performed using biotin-labeled lncSHRG or anti-sense control. Eluted fractions were resolved by SDS-PAGE, followed by silver staining and mass spectrometry. SATB1 was identified as a potential interactive protein of lncSHRG. (B) HCC sample lysates were incubated with anti-SATB1 at 4°C for 4 h, followed by an RNA immunoprecipitation assay. (C) Interaction of His-SATB1 with biotin-lncSHRG was checked by RNA pulldown assays. (D) lncSHRG (nt200∼400) was essential for the interaction with SATB1 as shown by domain mapping and RNA pulldown assays. (E) Biotin-labeled lncSHRG (nt200∼400) probe was incubated with His-SATB1 protein, followed by EMSA assays. (F) LncSHRG (nt200∼400) contributed to cell proliferation by MTT assays. Full-length lncSHRG but not truncation version (deletion of nt200∼400) promotes cell proliferation in HCC samples. (G) LncSHRG (nt200∼400) promoted colony formation. Full-length lncSHRG but not truncation version (deletion of nt200∼400) promotes colony formation of HCC sample cells. *p

Techniques Used: Labeling, SDS Page, Silver Staining, Mass Spectrometry, Incubation, Immunoprecipitation, MTT Assay

LncSHRG is highly expressed in HCC (A) Analysis of lncSHRG expression in peritumor and tumor tissues according to the microarray data in Wang’s cohort (GSE14520). (B) 30 pairs of peritumor and tumor HCC samples were collected. Then LncSHRG expression levels were analyzed in these sample tissues by RT-qPCR. Fold changes were normalized to endogenous ACTB . ( C) LncSHRG expression in peritumor and tumor tissues of sample #1 and #8 was checked by RNA hybridization in situ with biotin-labeled lncSHRG probes. Scale bars, 100μm. (D) Total RNAs were extracted from peritumor and HCC samples. LncSHRG and 18S rRNA (loading control) was examined by Northern blot. P: peritumor; T: tumor. (E) Total RNAs were extracted from indicative human HCC cell lines and lncSHRG expression was checked by RT-qPCR. Fold changes were normalized to endogenous ACTB . (F) Higher expression of lncSHRG in HCC samples. LncSHRG expression was analyzed with R language and Bioconductor according to the microarray data in Wang’s cohort (GSE54238). NL: normal livers; IL: chronic inflammatory livers; CL: cirrhotic livers; eHCC: early HCC; aHCC: advanced HCC. (G-I) LncSHRG expression levels were positively correlated with clinical stages and poor prognosis by expression analysis (G) and Kaplan–Meier survival analysis (H and I) according to the microarray data in Li’s cohort (GSE40144) and Wang’s cohort (GSE14520). * p
Figure Legend Snippet: LncSHRG is highly expressed in HCC (A) Analysis of lncSHRG expression in peritumor and tumor tissues according to the microarray data in Wang’s cohort (GSE14520). (B) 30 pairs of peritumor and tumor HCC samples were collected. Then LncSHRG expression levels were analyzed in these sample tissues by RT-qPCR. Fold changes were normalized to endogenous ACTB . ( C) LncSHRG expression in peritumor and tumor tissues of sample #1 and #8 was checked by RNA hybridization in situ with biotin-labeled lncSHRG probes. Scale bars, 100μm. (D) Total RNAs were extracted from peritumor and HCC samples. LncSHRG and 18S rRNA (loading control) was examined by Northern blot. P: peritumor; T: tumor. (E) Total RNAs were extracted from indicative human HCC cell lines and lncSHRG expression was checked by RT-qPCR. Fold changes were normalized to endogenous ACTB . (F) Higher expression of lncSHRG in HCC samples. LncSHRG expression was analyzed with R language and Bioconductor according to the microarray data in Wang’s cohort (GSE54238). NL: normal livers; IL: chronic inflammatory livers; CL: cirrhotic livers; eHCC: early HCC; aHCC: advanced HCC. (G-I) LncSHRG expression levels were positively correlated with clinical stages and poor prognosis by expression analysis (G) and Kaplan–Meier survival analysis (H and I) according to the microarray data in Li’s cohort (GSE40144) and Wang’s cohort (GSE14520). * p

Techniques Used: Expressing, Microarray, Quantitative RT-PCR, Hybridization, In Situ, Labeling, Northern Blot

32) Product Images from "Lnc RNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination"

Article Title: Lnc RNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination

Journal: EMBO Reports

doi: 10.15252/embr.201643668

GAS 5 suppresses IRF 4 transcription by binding PRC 2 and inhibiting microglial M2 polarization RNA IP analysis of the binding between EZH2 and GAS5, n = 3 experiments. RNA pull‐down analysis of the binding between EZH2 and GAS5. Quantitative PCR analysis of M1 and M2 markers in microglia transduced with the EZH2i lentivirus versus the control, n ≥ 4 experiments. ChIP analysis of microglia transduced with the CtrlOE or GAS5OE lentivirus. A relatively high enrichment was detected on the IRF4 promoter regions in MG GAS5OE versus the control using the anti‐EZH2 antibody, n = 3 experiments. ChIRP analysis of the binding between the IRF4 promoter and GAS5, n = 3 experiments. IGF1 served as a negative control. ChIP analysis of microglia transduced with the CtrlOE/GAS5OE (F) or Ctrli/GAS5i (G) lentivirus on the IRF4 promoter regions using the anti‐H3K27me3 antibody, n = 3 experiments. Quantitative PCR analysis of IRF4 in microglia transduced with the GAS5OE (H), GAS5i (I) or EZH2i (J) lentivirus, n ≥ 3 experiments per group. Western blotting analysis of IRF4 in microglia transduced with the GAS5OE (K), GAS5i (L), or EZH2i (M) lentivirus, n ≥ 3 experiments per group. Data information: * P
Figure Legend Snippet: GAS 5 suppresses IRF 4 transcription by binding PRC 2 and inhibiting microglial M2 polarization RNA IP analysis of the binding between EZH2 and GAS5, n = 3 experiments. RNA pull‐down analysis of the binding between EZH2 and GAS5. Quantitative PCR analysis of M1 and M2 markers in microglia transduced with the EZH2i lentivirus versus the control, n ≥ 4 experiments. ChIP analysis of microglia transduced with the CtrlOE or GAS5OE lentivirus. A relatively high enrichment was detected on the IRF4 promoter regions in MG GAS5OE versus the control using the anti‐EZH2 antibody, n = 3 experiments. ChIRP analysis of the binding between the IRF4 promoter and GAS5, n = 3 experiments. IGF1 served as a negative control. ChIP analysis of microglia transduced with the CtrlOE/GAS5OE (F) or Ctrli/GAS5i (G) lentivirus on the IRF4 promoter regions using the anti‐H3K27me3 antibody, n = 3 experiments. Quantitative PCR analysis of IRF4 in microglia transduced with the GAS5OE (H), GAS5i (I) or EZH2i (J) lentivirus, n ≥ 3 experiments per group. Western blotting analysis of IRF4 in microglia transduced with the GAS5OE (K), GAS5i (L), or EZH2i (M) lentivirus, n ≥ 3 experiments per group. Data information: * P

Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Transduction, Chromatin Immunoprecipitation, Negative Control, Western Blot

Analysis of the mechanism regulating microglial polarization by GAS 5 Silver staining to identify specific binding partners of GAS5 after RNA pull‐down experiment. RNA IP analysis between GR and GAS5. N = 3 experiments. RNA IP analysis between RbAp48 and GAS5. N = 3 experiments. RNA IP analysis between RING1A/B and GAS5. N = 3 experiments. ChIP analysis of microglia transduced with the CtrlOE or GAS5OE lentivirus. The promoter regions of STAT6 (E), PPARγ (F) and CEBP/β (G) were detected in MG GAS5OE versus the control using the anti‐EZH2 antibody. Data information: *** P
Figure Legend Snippet: Analysis of the mechanism regulating microglial polarization by GAS 5 Silver staining to identify specific binding partners of GAS5 after RNA pull‐down experiment. RNA IP analysis between GR and GAS5. N = 3 experiments. RNA IP analysis between RbAp48 and GAS5. N = 3 experiments. RNA IP analysis between RING1A/B and GAS5. N = 3 experiments. ChIP analysis of microglia transduced with the CtrlOE or GAS5OE lentivirus. The promoter regions of STAT6 (E), PPARγ (F) and CEBP/β (G) were detected in MG GAS5OE versus the control using the anti‐EZH2 antibody. Data information: *** P

Techniques Used: Silver Staining, Binding Assay, Chromatin Immunoprecipitation, Transduction

33) Product Images from "Helicobacter pylori infection related long noncoding RNA (lncRNA) AF147447 inhibits gastric cancer proliferation and invasion by targeting MUC2 and up-regulating miR-34c"

Article Title: Helicobacter pylori infection related long noncoding RNA (lncRNA) AF147447 inhibits gastric cancer proliferation and invasion by targeting MUC2 and up-regulating miR-34c

Journal: Oncotarget

doi: 10.18632/oncotarget.13165

LncRNA AF147447 physically associates with E2F1 ( A ) RIP experiments were performed using the E2F1 antibody to immunoprecipitate (IP) and a primer to detect lncRNA-AF147447. RIP enrichment was determined as RNA associated with E2F1 IP relative to an input control. ( B ) Diagram of lncRNA-AF147447 promoter region constructs. Luciferase reporter assays in 7901 cells, with co-transfection of lncRNA-AF147447, with siRNA-E2F1 or siRNA-control. ( C ) LncRNA-AF147447 expression was detected by qRT-PCR after cells transfecting with si-E2F1 or pcDNA-E2F1. ( D ) MUC2 mRNA and protein were detected by qRT-PCR and western blot. (* p
Figure Legend Snippet: LncRNA AF147447 physically associates with E2F1 ( A ) RIP experiments were performed using the E2F1 antibody to immunoprecipitate (IP) and a primer to detect lncRNA-AF147447. RIP enrichment was determined as RNA associated with E2F1 IP relative to an input control. ( B ) Diagram of lncRNA-AF147447 promoter region constructs. Luciferase reporter assays in 7901 cells, with co-transfection of lncRNA-AF147447, with siRNA-E2F1 or siRNA-control. ( C ) LncRNA-AF147447 expression was detected by qRT-PCR after cells transfecting with si-E2F1 or pcDNA-E2F1. ( D ) MUC2 mRNA and protein were detected by qRT-PCR and western blot. (* p

Techniques Used: Construct, Luciferase, Cotransfection, Expressing, Quantitative RT-PCR, Western Blot

Identification and validation of lncRNA-AF147447 targets ( A ) Biotinylated lncRNA-AF147447 or antisense RNA were incubated with nuclear extracts, targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of MUC2 and lncRNA-AF147447. Another MUC family: MUC1 is shown as a control. ( B ) Diagram of MUC2 promoter region constructs. ( C ) Luciferase reporter assays in 7901 cells, with co-transfection of wt or mt and lncRNA as indicated. ( D ) MUC2 expression was validated by qRT-PCR after transfecting with pLV-AF147447 or siRNA or their respective controls. ( E ) MUC2 expression was validated by western blot after transfecting with pLV-AF147447 or siRNA or their respective controls. (* p
Figure Legend Snippet: Identification and validation of lncRNA-AF147447 targets ( A ) Biotinylated lncRNA-AF147447 or antisense RNA were incubated with nuclear extracts, targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of MUC2 and lncRNA-AF147447. Another MUC family: MUC1 is shown as a control. ( B ) Diagram of MUC2 promoter region constructs. ( C ) Luciferase reporter assays in 7901 cells, with co-transfection of wt or mt and lncRNA as indicated. ( D ) MUC2 expression was validated by qRT-PCR after transfecting with pLV-AF147447 or siRNA or their respective controls. ( E ) MUC2 expression was validated by western blot after transfecting with pLV-AF147447 or siRNA or their respective controls. (* p

Techniques Used: Incubation, Western Blot, Construct, Luciferase, Cotransfection, Expressing, Quantitative RT-PCR

34) Product Images from "Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) Promotes Hepatocellular Carcinoma Progression Through Interaction with High-Mobility Group AT-hook 2 and MiR-4516"

Article Title: Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) Promotes Hepatocellular Carcinoma Progression Through Interaction with High-Mobility Group AT-hook 2 and MiR-4516

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.911179

LSINCT5 acts as a ceRNA of miR-4516 to promote HCC progression. ( A ) RIP assays followed by qPCR to identify putative miRNAs associated with LSINCT5. ( B ) The miRNAs were verified by RNA pulldown a with biotin-labeled sense or antisense LSINCT5. ( C ) Luciferase reporter assays identified miR-4516 could interact with LSINCT5 (top). RNA pulldown assay with anti-AGO2 was performed in HepG2 cells with miR-4516 overexpression followed by qPCR to enrich LSINCT5 (bottom). ( D ) The predicted base-pairing between LSINCT5 and miR-4516 at miRDB. Three hits were detected. ( E ) RNA-FISH assay revealed the co-localization of LSINCT5 (Alexa Fluor 488, green dots) and miR-4516 (Cy3, red dots) in HepG2 cells. Scale bar, 5 μm. ( F ) CCK-8 viability assay for Huh7 cells and HepG2 cells transfected with plasmids as indicated. ( G ) Tumor sphere formation assay for HepG2 cells transfected with negative control (NC), miR-4516 mimics + oe Vec or miR-4516 mimics + LSINCT5. Quantification results were shown on the right panel. ( H ) Western blot assays for BclxL, STAT3, and pSTAT3 (Tyr705) expression in HepG2 cells by knocking down or overexpressing LSINCT5. CCK-8– cell counting Kit-8; ceRNA – competing endogenous RNA; FISH – fluorescent in situ hybridization; HCC – hepatocellular carcinoma; LSINCT5 – long stress induced non-coding transcripts 5; miRNAs – micro RNAs; oe – overexpression; RIP – RNA immunoprecipitation.
Figure Legend Snippet: LSINCT5 acts as a ceRNA of miR-4516 to promote HCC progression. ( A ) RIP assays followed by qPCR to identify putative miRNAs associated with LSINCT5. ( B ) The miRNAs were verified by RNA pulldown a with biotin-labeled sense or antisense LSINCT5. ( C ) Luciferase reporter assays identified miR-4516 could interact with LSINCT5 (top). RNA pulldown assay with anti-AGO2 was performed in HepG2 cells with miR-4516 overexpression followed by qPCR to enrich LSINCT5 (bottom). ( D ) The predicted base-pairing between LSINCT5 and miR-4516 at miRDB. Three hits were detected. ( E ) RNA-FISH assay revealed the co-localization of LSINCT5 (Alexa Fluor 488, green dots) and miR-4516 (Cy3, red dots) in HepG2 cells. Scale bar, 5 μm. ( F ) CCK-8 viability assay for Huh7 cells and HepG2 cells transfected with plasmids as indicated. ( G ) Tumor sphere formation assay for HepG2 cells transfected with negative control (NC), miR-4516 mimics + oe Vec or miR-4516 mimics + LSINCT5. Quantification results were shown on the right panel. ( H ) Western blot assays for BclxL, STAT3, and pSTAT3 (Tyr705) expression in HepG2 cells by knocking down or overexpressing LSINCT5. CCK-8– cell counting Kit-8; ceRNA – competing endogenous RNA; FISH – fluorescent in situ hybridization; HCC – hepatocellular carcinoma; LSINCT5 – long stress induced non-coding transcripts 5; miRNAs – micro RNAs; oe – overexpression; RIP – RNA immunoprecipitation.

Techniques Used: Real-time Polymerase Chain Reaction, Labeling, Luciferase, Over Expression, Fluorescence In Situ Hybridization, CCK-8 Assay, Viability Assay, Transfection, Tube Formation Assay, Negative Control, Western Blot, Expressing, Cell Counting, In Situ Hybridization, Immunoprecipitation

35) Product Images from "The long noncoding RNA PCGEM1 promotes cell proliferation, migration and invasion via targeting the miR-182/FBXW11 axis in cervical cancer"

Article Title: The long noncoding RNA PCGEM1 promotes cell proliferation, migration and invasion via targeting the miR-182/FBXW11 axis in cervical cancer

Journal: Cancer Cell International

doi: 10.1186/s12935-019-1030-8

PCGEM1 directly interacts with miR-182. a Cytoplasmic and nuclear RNA fraction assay showed the cellular location of PCGEM1 in CC cells. U6 RNA served as a positive control for nuclear gene expression. GAPDH mRNA served as a positive control for cytoplasmic gene expression. b MS2-RIP followed by qRT-PCR was conducted to detect endogenous microRNAs associated with lncRNA PCGEM1. c HeLa and SiHa cell lysates were incubated with biotin-labeled wild-type or mutant PCGEM1; after pull-down, microRNAs were extracted, and the amount of miR-182 was assessed by qRT-PCR. d HeLa and SiHa cells were cotransfected with miR-182 mimics and luciferase reporters containing wild-type or mutant PCGEM1. After 48 h, luciferase activity was measured and presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. e Anti-AGO2 RIP was performed in HeLa and SiHa cells transfected with miR-NC or miR-182, followed by qRT-PCR to detect PCGEM1 associated with AGO2. f SiHa cells were transfected with wild-type or mutant PCGEM1. After 48 h, the miR-182 expression was assessed by qRT-PCR. g HeLa cells were transfected with PCGEM1 siRNAs. After 48 h, the miR-182 expression was assessed by qRT-PCR. h The expression of miR-182 in 68 pairs of CC and normal cervical tissues was determined by qRT-PCR. i Expression levels of PCGEM1 and miR-182 were subjected to Pearson correlation analysis. *p
Figure Legend Snippet: PCGEM1 directly interacts with miR-182. a Cytoplasmic and nuclear RNA fraction assay showed the cellular location of PCGEM1 in CC cells. U6 RNA served as a positive control for nuclear gene expression. GAPDH mRNA served as a positive control for cytoplasmic gene expression. b MS2-RIP followed by qRT-PCR was conducted to detect endogenous microRNAs associated with lncRNA PCGEM1. c HeLa and SiHa cell lysates were incubated with biotin-labeled wild-type or mutant PCGEM1; after pull-down, microRNAs were extracted, and the amount of miR-182 was assessed by qRT-PCR. d HeLa and SiHa cells were cotransfected with miR-182 mimics and luciferase reporters containing wild-type or mutant PCGEM1. After 48 h, luciferase activity was measured and presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. e Anti-AGO2 RIP was performed in HeLa and SiHa cells transfected with miR-NC or miR-182, followed by qRT-PCR to detect PCGEM1 associated with AGO2. f SiHa cells were transfected with wild-type or mutant PCGEM1. After 48 h, the miR-182 expression was assessed by qRT-PCR. g HeLa cells were transfected with PCGEM1 siRNAs. After 48 h, the miR-182 expression was assessed by qRT-PCR. h The expression of miR-182 in 68 pairs of CC and normal cervical tissues was determined by qRT-PCR. i Expression levels of PCGEM1 and miR-182 were subjected to Pearson correlation analysis. *p

Techniques Used: Positive Control, Expressing, Quantitative RT-PCR, Incubation, Labeling, Mutagenesis, Luciferase, Activity Assay, Transfection

36) Product Images from "LncKdm2b controls self‐renewal of embryonic stem cells via activating expression of transcription factor Zbtb3"

Article Title: LncKdm2b controls self‐renewal of embryonic stem cells via activating expression of transcription factor Zbtb3

Journal: The EMBO Journal

doi: 10.15252/embj.201797174

LncKdm2b promotes the ATPase activity of SRCAP A Biotin RNA pull‐downs were performed with nuclear extracts of mouse ESCs using full‐length lncKdm2b transcript (Sense), antisense, and Xist A repeats sequence control followed with mass spectrometry. B, C The interaction of SRCAP with lncKdm2b was confirmed by immunoblotting (B) and CHIRP assay (C). Biotinylated probes were hybridized to lncKdm2b . D Pluripotent ESCs and E3.5 blastocysts were probed with lncKdm2b by RNA‐FISH, followed by immunofluorescence staining for SRCAP. Green: lncKdm2b probe; red: SRCAP; nuclei were counterstained by DAPI. Scale bar, 10 μm. For normal ESC clone, n = 240; for LIF‐withdrawal ESC clone, n = 130; for lncKdm2b −/− ESC clone, n = 105; for E3.5 embryos, n = 119. E Interaction of lncKdm2b with SRCAP was verified by RIP assay. ESC lysates were incubated with anti‐SRCAP antibody, followed by RNA immunoprecipitation (RIP) assay. RNA was extracted and reversely transcribed. LncKdm2b . F Full‐length and truncated fragments of lncKdm2b were in vitro ‐transcribed to biotin‐labeled RNA followed with RNA pull‐down and immunoblotting. d450–700 denotes the truncated fragment of lncKdm2b deleting nt 450–700. d700–1,000 denotes the truncated fragment of lncKdm2b deleting nt 700–1,000. G Nuclear extracts of ESCs and biotin‐labeled lncKdm2b (450–700 nt) probes were incubated for EMSA assays. Anti‐SRCAP antibody was preincubated with nuclear extracts that caused supershift. H ESC lysates were immunoprecipitated with anti‐SRCAP antibody, followed by detection of ATPase activities. Biotin‐labeled lncKdm2b and lncKdm2b (nt 450–700) fragments were generated by in vitro transcription by T7 RNA polymerase. Mouse IgG IP was used as a background control. Relative OD values were normalized to IgG background control and shown as fold changes as means ± SD. lnc, lncKdm2b ; oe, overexpression. ** P = 0.0034, ** P = 0.0089, ** P = 0.0023, ** P = 0.0011 by unpaired Student's t ‐test. I lncKdm2b +/+ and lncKdm2b −/− ESC cell lysates were immunoprecipitated with anti‐SRCAP antibody, followed by immunoblotting with the indicated antibodies. Data information: All data are representative of five independent experiments.
Figure Legend Snippet: LncKdm2b promotes the ATPase activity of SRCAP A Biotin RNA pull‐downs were performed with nuclear extracts of mouse ESCs using full‐length lncKdm2b transcript (Sense), antisense, and Xist A repeats sequence control followed with mass spectrometry. B, C The interaction of SRCAP with lncKdm2b was confirmed by immunoblotting (B) and CHIRP assay (C). Biotinylated probes were hybridized to lncKdm2b . D Pluripotent ESCs and E3.5 blastocysts were probed with lncKdm2b by RNA‐FISH, followed by immunofluorescence staining for SRCAP. Green: lncKdm2b probe; red: SRCAP; nuclei were counterstained by DAPI. Scale bar, 10 μm. For normal ESC clone, n = 240; for LIF‐withdrawal ESC clone, n = 130; for lncKdm2b −/− ESC clone, n = 105; for E3.5 embryos, n = 119. E Interaction of lncKdm2b with SRCAP was verified by RIP assay. ESC lysates were incubated with anti‐SRCAP antibody, followed by RNA immunoprecipitation (RIP) assay. RNA was extracted and reversely transcribed. LncKdm2b . F Full‐length and truncated fragments of lncKdm2b were in vitro ‐transcribed to biotin‐labeled RNA followed with RNA pull‐down and immunoblotting. d450–700 denotes the truncated fragment of lncKdm2b deleting nt 450–700. d700–1,000 denotes the truncated fragment of lncKdm2b deleting nt 700–1,000. G Nuclear extracts of ESCs and biotin‐labeled lncKdm2b (450–700 nt) probes were incubated for EMSA assays. Anti‐SRCAP antibody was preincubated with nuclear extracts that caused supershift. H ESC lysates were immunoprecipitated with anti‐SRCAP antibody, followed by detection of ATPase activities. Biotin‐labeled lncKdm2b and lncKdm2b (nt 450–700) fragments were generated by in vitro transcription by T7 RNA polymerase. Mouse IgG IP was used as a background control. Relative OD values were normalized to IgG background control and shown as fold changes as means ± SD. lnc, lncKdm2b ; oe, overexpression. ** P = 0.0034, ** P = 0.0089, ** P = 0.0023, ** P = 0.0011 by unpaired Student's t ‐test. I lncKdm2b +/+ and lncKdm2b −/− ESC cell lysates were immunoprecipitated with anti‐SRCAP antibody, followed by immunoblotting with the indicated antibodies. Data information: All data are representative of five independent experiments.

Techniques Used: Activity Assay, Sequencing, Mass Spectrometry, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Incubation, Immunoprecipitation, In Vitro, Labeling, Generated, Over Expression

37) Product Images from "Ets-1 promoter-associated noncoding RNA regulates the NONO/ERG/Ets-1 axis to drive gastric cancer progression"

Article Title: Ets-1 promoter-associated noncoding RNA regulates the NONO/ERG/Ets-1 axis to drive gastric cancer progression

Journal: Oncogene

doi: 10.1038/s41388-018-0302-4

pancEts-1 is a lncRNA associated with poor survival of gastric cancer. a Scheme indicating the existence of pancEts-1 transcribed upstream the Ets-1 promoter region. b RNA fluorescence in situ hybridization images showing the nuclear and cytoplasmic localization of pancEts-1 in MKN-45 cells using a 138-bp antisense probe (red), with the nuclei staining by DAPI (blue). Sense probe and antisense probe with RNase A (20 μg) treatment were used as negative controls. Scale bars: 10 μm. c Real-time qRT-PCR assay revealing the pancEts-1 transcript levels (normalized to β-actin) in normal gastric mucosa ( n = 30) and cultured gastric cancer cell lines (mean ± SD, n = 5). d Real-time qRT-PCR assay indicating the differential expression of pancEts-1 transcript (normalized to β-actin) in normal gastric mucosa ( n = 30) and gastric cancer tissues ( n = 81). e , f Real-time qRT-PCR assay showing the pancEts-1 transcript levels (normalized to β-actin) in gastric cancer tissues with differential status of metastasis ( e ) or Ets-1 immunostaining ( f ). g The positive correlation between pancEts-1 and Ets-1 transcript levels in gastric cancer tissues ( n = 81). h Kaplan–Meier curves indicating overall survival of 81 gastric cancer patients (cutoff value = 2.833) and overall (OS) and first progression (FP) survival of those derived from Kaplan-Meier plotter with low or high pancEts-1 expression (cutoff values = 14.0 and 14.0). Student’s t test compared gene expression levels in c – f . Pearson’s correlation coefficient analysis in g . Log-rank test for survival comparison in h
Figure Legend Snippet: pancEts-1 is a lncRNA associated with poor survival of gastric cancer. a Scheme indicating the existence of pancEts-1 transcribed upstream the Ets-1 promoter region. b RNA fluorescence in situ hybridization images showing the nuclear and cytoplasmic localization of pancEts-1 in MKN-45 cells using a 138-bp antisense probe (red), with the nuclei staining by DAPI (blue). Sense probe and antisense probe with RNase A (20 μg) treatment were used as negative controls. Scale bars: 10 μm. c Real-time qRT-PCR assay revealing the pancEts-1 transcript levels (normalized to β-actin) in normal gastric mucosa ( n = 30) and cultured gastric cancer cell lines (mean ± SD, n = 5). d Real-time qRT-PCR assay indicating the differential expression of pancEts-1 transcript (normalized to β-actin) in normal gastric mucosa ( n = 30) and gastric cancer tissues ( n = 81). e , f Real-time qRT-PCR assay showing the pancEts-1 transcript levels (normalized to β-actin) in gastric cancer tissues with differential status of metastasis ( e ) or Ets-1 immunostaining ( f ). g The positive correlation between pancEts-1 and Ets-1 transcript levels in gastric cancer tissues ( n = 81). h Kaplan–Meier curves indicating overall survival of 81 gastric cancer patients (cutoff value = 2.833) and overall (OS) and first progression (FP) survival of those derived from Kaplan-Meier plotter with low or high pancEts-1 expression (cutoff values = 14.0 and 14.0). Student’s t test compared gene expression levels in c – f . Pearson’s correlation coefficient analysis in g . Log-rank test for survival comparison in h

Techniques Used: Fluorescence, In Situ Hybridization, Staining, Quantitative RT-PCR, Cell Culture, Expressing, Immunostaining, Derivative Assay

pancEts-1 regulates Ets-1 expression by facilitating NONO-mediated ERG transactivation. a IP, Commassie blue staining (left) and mass spectrometry (MS) assay (right) showing the changes in NONO interacting proteins in NCI-N87 cells stably transfected with empty vector (mock) or pancEts-1 . b IP and western blot revealing the endogenous interaction between NONO and ERG in the NCI-N87 and AGS cells, c , d IP and western blot indicating the interaction between NONO and ERG in NCI-N87 cells transfected with HA-tagged ERG or FLAG-tagged NONO truncates. e IP and western blot showing the interaction between NONO and ERG in NCI-N87 and MKN-45 cells stably transfected with mock, pancEts-1 , scramble shRNA (sh-RNA), or sh-pancEts-1 #2. f ChIP and qPCR assays indicating the binding of ERG to Ets-1 promoter in gastric cancer cells, and its changes in those stably transfected with mock, NONO , pancEts-1 , sh-Scb, sh-NONO #2, or sh-pancEts-1 #2 (mean ± SD, n = 4). g , h Dual-luciferase ( g ) and western blot ( h ) assays showing the promoter activity and expression of Ets-1 in gastric cancer cells, and their changes in those stably transfected with mock, ERG , pancEts-1 , sh-Scb, sh-ERG #1, sh-ERG #2, or sh-pancEts-1 #2 (mean ± SD, n = 4). Student’s t test analyzed the difference in f and g . * P
Figure Legend Snippet: pancEts-1 regulates Ets-1 expression by facilitating NONO-mediated ERG transactivation. a IP, Commassie blue staining (left) and mass spectrometry (MS) assay (right) showing the changes in NONO interacting proteins in NCI-N87 cells stably transfected with empty vector (mock) or pancEts-1 . b IP and western blot revealing the endogenous interaction between NONO and ERG in the NCI-N87 and AGS cells, c , d IP and western blot indicating the interaction between NONO and ERG in NCI-N87 cells transfected with HA-tagged ERG or FLAG-tagged NONO truncates. e IP and western blot showing the interaction between NONO and ERG in NCI-N87 and MKN-45 cells stably transfected with mock, pancEts-1 , scramble shRNA (sh-RNA), or sh-pancEts-1 #2. f ChIP and qPCR assays indicating the binding of ERG to Ets-1 promoter in gastric cancer cells, and its changes in those stably transfected with mock, NONO , pancEts-1 , sh-Scb, sh-NONO #2, or sh-pancEts-1 #2 (mean ± SD, n = 4). g , h Dual-luciferase ( g ) and western blot ( h ) assays showing the promoter activity and expression of Ets-1 in gastric cancer cells, and their changes in those stably transfected with mock, ERG , pancEts-1 , sh-Scb, sh-ERG #1, sh-ERG #2, or sh-pancEts-1 #2 (mean ± SD, n = 4). Student’s t test analyzed the difference in f and g . * P

Techniques Used: Expressing, Staining, Mass Spectrometry, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Luciferase, Activity Assay

pancEts-1 interacts with NONO protein in gastric cancer cells. a Biotin-labeled RNA pull-down (left) and mass spectrometry (MS) assay (right) showing the interaction between pancEts-1 and NONO protein in MKN-45 cells. The pancEts-1 antisense (AS)- and bead-bound protein served as negative controls. b RIP assay using NONO antibody indicating the interaction between pancEts-1 and NONO protein in NCI-N87 cells transfected with a series of pancEts-1 truncates. The IgG-bound RNA was taken as negative control. c , d , Western blot assay depicting the recovered NONO levels of cellular nuclear extracts pulled down by biotin-labeled pancEts-1 truncates. e In vitro binding assay showing the recovered pancEts-1 levels by RIP (lower) after incubation with full-length (1–471 amino acids), ΔN (75–471 amino acids), ΔC (1–374 amino acids), Drosophila behavior/human splicing (DBHS, 75–374 amino acids), ΔRRM1 (149–374 amino acids), ΔRRM1 + 2 (231–374 amino acids), or coiled-coil (269–374 amino acids) of GST-tagged recombinant NONO protein validated by western blot (upper). f RNA EMSA determining the interaction between recombinant or endogenous NONO protein and biotin-labeled RNA probes for pancEts-1 (arrowhead), with or without competition using an excess of unlabeled homologous RNA probe or treatment using NONO antibody.
Figure Legend Snippet: pancEts-1 interacts with NONO protein in gastric cancer cells. a Biotin-labeled RNA pull-down (left) and mass spectrometry (MS) assay (right) showing the interaction between pancEts-1 and NONO protein in MKN-45 cells. The pancEts-1 antisense (AS)- and bead-bound protein served as negative controls. b RIP assay using NONO antibody indicating the interaction between pancEts-1 and NONO protein in NCI-N87 cells transfected with a series of pancEts-1 truncates. The IgG-bound RNA was taken as negative control. c , d , Western blot assay depicting the recovered NONO levels of cellular nuclear extracts pulled down by biotin-labeled pancEts-1 truncates. e In vitro binding assay showing the recovered pancEts-1 levels by RIP (lower) after incubation with full-length (1–471 amino acids), ΔN (75–471 amino acids), ΔC (1–374 amino acids), Drosophila behavior/human splicing (DBHS, 75–374 amino acids), ΔRRM1 (149–374 amino acids), ΔRRM1 + 2 (231–374 amino acids), or coiled-coil (269–374 amino acids) of GST-tagged recombinant NONO protein validated by western blot (upper). f RNA EMSA determining the interaction between recombinant or endogenous NONO protein and biotin-labeled RNA probes for pancEts-1 (arrowhead), with or without competition using an excess of unlabeled homologous RNA probe or treatment using NONO antibody.

Techniques Used: Labeling, Mass Spectrometry, Transfection, Negative Control, Western Blot, In Vitro, Binding Assay, Incubation, Recombinant

38) Product Images from "LncRNA GAS5 enhanced the killing effect of NK cell on liver cancer through regulating miR-544/RUNX3"

Article Title: LncRNA GAS5 enhanced the killing effect of NK cell on liver cancer through regulating miR-544/RUNX3

Journal: Innate Immunity

doi: 10.1177/1753425919827632

Regulation role of lncRNA GAS5 in miR-544. (a) Bioinformatics software predicted binding sites between GAS5 and miR-544. (b) AGO2 Ab was used for RNA immunoprecipitation. AGO2 protein level was detected by IP-Western, and GAS5 and miR-544 were detected by qRT-PCR. GAS5 and miR-544 were enriched in AGO2. (c) AGO2 in GAS5 pull-down complex was detected by Western blot, and miR-544 enrichment in GAS5 pull-down complex was detected by qRT-PCR. Loc, negative control of GAS5 pull-down complex. (d) NK92 cells were transfected with lenti-NC or lenti-GAS5. Compared with the lenti-NC group, miR-544 level was significantly decreased in lenti-GAS5 group. * P
Figure Legend Snippet: Regulation role of lncRNA GAS5 in miR-544. (a) Bioinformatics software predicted binding sites between GAS5 and miR-544. (b) AGO2 Ab was used for RNA immunoprecipitation. AGO2 protein level was detected by IP-Western, and GAS5 and miR-544 were detected by qRT-PCR. GAS5 and miR-544 were enriched in AGO2. (c) AGO2 in GAS5 pull-down complex was detected by Western blot, and miR-544 enrichment in GAS5 pull-down complex was detected by qRT-PCR. Loc, negative control of GAS5 pull-down complex. (d) NK92 cells were transfected with lenti-NC or lenti-GAS5. Compared with the lenti-NC group, miR-544 level was significantly decreased in lenti-GAS5 group. * P

Techniques Used: Software, Binding Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Negative Control, Transfection

39) Product Images from "LINC01006 promotes cell proliferation and metastasis in pancreatic cancer via miR-2682-5p/HOXB8 axis"

Article Title: LINC01006 promotes cell proliferation and metastasis in pancreatic cancer via miR-2682-5p/HOXB8 axis

Journal: Cancer Cell International

doi: 10.1186/s12935-019-1036-2

LINC01006 serves as a sponge of miR-2682-5p in PC. a Subcellular fractionation was utilized to detect the distribution of LINC01006 in nuclear and cytoplasm. b Western blot assay was employed to confirm the combination between LINC01006 and Ago2. c MiRNAs that could bind with LINC01006 were obtained by referring to starbase. d The binding ability among LINC01006 and miRNAs was examined by RNA pull down assay. e The expression of miR-2682-5p in PC cell lines and normal human pancreatic duct epithelial cell line was detected by RT-qPCR. f RT-qPCR assays were used to detect the expression of miR-2682-5p in transfected cells. g RIP assay was carried out to verify that LINC01006 could bind with miR-2682-5p. h RT-qPCR assay analyzed the overexpression efficiency of miR-2682-5p in PANC-1 and BxPC-3 cells. i The binding site between LINC01006 and miR-2682-5p was obtained from starBase, which was then verified by luciferase reporter assay. *P
Figure Legend Snippet: LINC01006 serves as a sponge of miR-2682-5p in PC. a Subcellular fractionation was utilized to detect the distribution of LINC01006 in nuclear and cytoplasm. b Western blot assay was employed to confirm the combination between LINC01006 and Ago2. c MiRNAs that could bind with LINC01006 were obtained by referring to starbase. d The binding ability among LINC01006 and miRNAs was examined by RNA pull down assay. e The expression of miR-2682-5p in PC cell lines and normal human pancreatic duct epithelial cell line was detected by RT-qPCR. f RT-qPCR assays were used to detect the expression of miR-2682-5p in transfected cells. g RIP assay was carried out to verify that LINC01006 could bind with miR-2682-5p. h RT-qPCR assay analyzed the overexpression efficiency of miR-2682-5p in PANC-1 and BxPC-3 cells. i The binding site between LINC01006 and miR-2682-5p was obtained from starBase, which was then verified by luciferase reporter assay. *P

Techniques Used: Fractionation, Western Blot, Binding Assay, Pull Down Assay, Expressing, Quantitative RT-PCR, Transfection, Over Expression, Luciferase, Reporter Assay

40) Product Images from "Therapeutic targeting of circ‐CUX1/ EWSR1/ MAZ axis inhibits glycolysis and neuroblastoma progression"

Article Title: Therapeutic targeting of circ‐CUX1/ EWSR1/ MAZ axis inhibits glycolysis and neuroblastoma progression

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201910835

Circ‐CUX1 facilitates EWSR1‐mediated MAZ transactivation in NB cells Volcano plots indicating RNA‐seq results of 781 up‐regulated and 434 down‐regulated genes in IMR32 cells upon stable circ‐CUX1 over‐expression (fold change > 1.5, P
Figure Legend Snippet: Circ‐CUX1 facilitates EWSR1‐mediated MAZ transactivation in NB cells Volcano plots indicating RNA‐seq results of 781 up‐regulated and 434 down‐regulated genes in IMR32 cells upon stable circ‐CUX1 over‐expression (fold change > 1.5, P

Techniques Used: RNA Sequencing Assay, Over Expression

Related Articles

Negative Control:

Article Title: Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma
Article Snippet: .. RNA pull-down MUC5B-AS1 and lncRNA-SFTA1P (negative control) were in vitro transcribed, respectively, from vector pcDNA3.1-MUC5B-AS1 and pcDNA3.1-SFTA1P, and biotin-labeled with the Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Promega, Madison, WI, USA). .. The biotin-labeled RNA were purified with an RNeasy Mini Kit (Qiagen).

Pull Down Assay:

Article Title: Long noncoding RNA GAPLINC promotes invasion in colorectal cancer by targeting SNAI2 through binding with PSF and NONO
Article Snippet: .. RNA pull-down assay and mass spectrometry The biotin-labeled RNAs of GAPLINC were in vitro transcribed using Biotin RNA Labeling Mix (Roche, CA, USA) and T7 RNA polymerase (New England Biolabs, MA, USA), and purified using the EZNA RNA Probe Purification Kit (Omega, GA, USA). .. The protein lysate from 107 HCT116 cells were incubated with 2 μg of biotinylated RNA and mixed with T1 beads (Invitrogen, CA, USA).

Labeling:

Article Title: Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer
Article Snippet: .. Briefly, biotin-labeled RNAs were in vitro transcribed with the Biotin RNA Labeling Mix (RocheDiagnostics, Indianapolis, IN) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). .. Cell nuclear proteins were extracted using the ProteoJETTM Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas, St. Leon-Rot, Germany).

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3
Article Snippet: .. Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). .. Next, whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C.

Article Title: Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma
Article Snippet: .. RNA pull-down MUC5B-AS1 and lncRNA-SFTA1P (negative control) were in vitro transcribed, respectively, from vector pcDNA3.1-MUC5B-AS1 and pcDNA3.1-SFTA1P, and biotin-labeled with the Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Promega, Madison, WI, USA). .. The biotin-labeled RNA were purified with an RNeasy Mini Kit (Qiagen).

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3
Article Snippet: .. RNA pulldown Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). .. Next, whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C.

Article Title: Role of the ATPase/helicase maleless (MLE) in the assembly, targeting, spreading and function of the male-specific lethal (MSL) complex of Drosophila
Article Snippet: .. Briefly, single-stranded antisense roX1 and roX 2 probes were labeled by in vitro transcription with a biotin RNA labeling mix (RocheApplied Science, Indianapolis, IN, USA) with 1.6 kb roX1 (nucleotides 1536 to 3110) or 0.8 kb roX2 cDNA fragments in pTopo 2.1 vector as the template. ..

Article Title: Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a
Article Snippet: .. In vitro biotin-labeled RNAs (lncAVAN and its antisense RNA) were transcribed with biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Promega) and purified using the RNeasy Mini Kit (QIAGEN). .. Biotinylated RNA was incubated with cell lysate, and precipitated proteins were separated via SDS-PAGE.

Article Title: Long noncoding RNA GAPLINC promotes invasion in colorectal cancer by targeting SNAI2 through binding with PSF and NONO
Article Snippet: .. RNA pull-down assay and mass spectrometry The biotin-labeled RNAs of GAPLINC were in vitro transcribed using Biotin RNA Labeling Mix (Roche, CA, USA) and T7 RNA polymerase (New England Biolabs, MA, USA), and purified using the EZNA RNA Probe Purification Kit (Omega, GA, USA). .. The protein lysate from 107 HCT116 cells were incubated with 2 μg of biotinylated RNA and mixed with T1 beads (Invitrogen, CA, USA).

Article Title: Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica
Article Snippet: .. These molecules and RNA molecules selected from the seventh round of the SELEX protocol (R7), were labeled with the Biotin RNA Labeling Mix (Roche). .. Their size and integrity were verified by agarose gel electrophoresis and chemiluminescence (Chemiluminescent Nucleic Acid Detection Module, Pierce).

Purification:

Article Title: Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer
Article Snippet: .. Briefly, biotin-labeled RNAs were in vitro transcribed with the Biotin RNA Labeling Mix (RocheDiagnostics, Indianapolis, IN) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). .. Cell nuclear proteins were extracted using the ProteoJETTM Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas, St. Leon-Rot, Germany).

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3
Article Snippet: .. Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). .. Next, whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C.

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3
Article Snippet: .. RNA pulldown Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). .. Next, whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C.

Article Title: Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a
Article Snippet: .. In vitro biotin-labeled RNAs (lncAVAN and its antisense RNA) were transcribed with biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Promega) and purified using the RNeasy Mini Kit (QIAGEN). .. Biotinylated RNA was incubated with cell lysate, and precipitated proteins were separated via SDS-PAGE.

Article Title: Long noncoding RNA GAPLINC promotes invasion in colorectal cancer by targeting SNAI2 through binding with PSF and NONO
Article Snippet: .. RNA pull-down assay and mass spectrometry The biotin-labeled RNAs of GAPLINC were in vitro transcribed using Biotin RNA Labeling Mix (Roche, CA, USA) and T7 RNA polymerase (New England Biolabs, MA, USA), and purified using the EZNA RNA Probe Purification Kit (Omega, GA, USA). .. The protein lysate from 107 HCT116 cells were incubated with 2 μg of biotinylated RNA and mixed with T1 beads (Invitrogen, CA, USA).

Mass Spectrometry:

Article Title: Long noncoding RNA GAPLINC promotes invasion in colorectal cancer by targeting SNAI2 through binding with PSF and NONO
Article Snippet: .. RNA pull-down assay and mass spectrometry The biotin-labeled RNAs of GAPLINC were in vitro transcribed using Biotin RNA Labeling Mix (Roche, CA, USA) and T7 RNA polymerase (New England Biolabs, MA, USA), and purified using the EZNA RNA Probe Purification Kit (Omega, GA, USA). .. The protein lysate from 107 HCT116 cells were incubated with 2 μg of biotinylated RNA and mixed with T1 beads (Invitrogen, CA, USA).

Plasmid Preparation:

Article Title: Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma
Article Snippet: .. RNA pull-down MUC5B-AS1 and lncRNA-SFTA1P (negative control) were in vitro transcribed, respectively, from vector pcDNA3.1-MUC5B-AS1 and pcDNA3.1-SFTA1P, and biotin-labeled with the Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Promega, Madison, WI, USA). .. The biotin-labeled RNA were purified with an RNeasy Mini Kit (Qiagen).

Article Title: Role of the ATPase/helicase maleless (MLE) in the assembly, targeting, spreading and function of the male-specific lethal (MSL) complex of Drosophila
Article Snippet: .. Briefly, single-stranded antisense roX1 and roX 2 probes were labeled by in vitro transcription with a biotin RNA labeling mix (RocheApplied Science, Indianapolis, IN, USA) with 1.6 kb roX1 (nucleotides 1536 to 3110) or 0.8 kb roX2 cDNA fragments in pTopo 2.1 vector as the template. ..

In Vitro:

Article Title: Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer
Article Snippet: .. Briefly, biotin-labeled RNAs were in vitro transcribed with the Biotin RNA Labeling Mix (RocheDiagnostics, Indianapolis, IN) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). .. Cell nuclear proteins were extracted using the ProteoJETTM Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas, St. Leon-Rot, Germany).

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3
Article Snippet: .. Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). .. Next, whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C.

Article Title: Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma
Article Snippet: .. RNA pull-down MUC5B-AS1 and lncRNA-SFTA1P (negative control) were in vitro transcribed, respectively, from vector pcDNA3.1-MUC5B-AS1 and pcDNA3.1-SFTA1P, and biotin-labeled with the Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Promega, Madison, WI, USA). .. The biotin-labeled RNA were purified with an RNeasy Mini Kit (Qiagen).

Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3
Article Snippet: .. RNA pulldown Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). .. Next, whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C.

Article Title: Role of the ATPase/helicase maleless (MLE) in the assembly, targeting, spreading and function of the male-specific lethal (MSL) complex of Drosophila
Article Snippet: .. Briefly, single-stranded antisense roX1 and roX 2 probes were labeled by in vitro transcription with a biotin RNA labeling mix (RocheApplied Science, Indianapolis, IN, USA) with 1.6 kb roX1 (nucleotides 1536 to 3110) or 0.8 kb roX2 cDNA fragments in pTopo 2.1 vector as the template. ..

Article Title: Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a
Article Snippet: .. In vitro biotin-labeled RNAs (lncAVAN and its antisense RNA) were transcribed with biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Promega) and purified using the RNeasy Mini Kit (QIAGEN). .. Biotinylated RNA was incubated with cell lysate, and precipitated proteins were separated via SDS-PAGE.

Article Title: Long noncoding RNA GAPLINC promotes invasion in colorectal cancer by targeting SNAI2 through binding with PSF and NONO
Article Snippet: .. RNA pull-down assay and mass spectrometry The biotin-labeled RNAs of GAPLINC were in vitro transcribed using Biotin RNA Labeling Mix (Roche, CA, USA) and T7 RNA polymerase (New England Biolabs, MA, USA), and purified using the EZNA RNA Probe Purification Kit (Omega, GA, USA). .. The protein lysate from 107 HCT116 cells were incubated with 2 μg of biotinylated RNA and mixed with T1 beads (Invitrogen, CA, USA).

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    Roche biotin rna labeling mix
    AVAN direct binds to TRIM25 and enhances the antivirus immune response (A) <t>RNA</t> pull-down of AVAN -associated proteins using biotinylated AVAN or <t>antisense</t> probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. (B) Pull-down western blot showing that AVAN can bind directly to TRIM25. (C) ChIRP followed by western blot show that AVAN can bind to TRIM25. (D and E) Exogenous (D) and endogenous (E) RIP of TRIM25 in BJ501 infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR. (F) AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD or Flag-Ring. (G) Truncated AVAN pull-down, truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pulldown. (H and I) TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN s or siRNAs, followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. (J and K) Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVAN s or siRNAs. Anti-RIG-I antibody was used for immunoprecipitated. (L) Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN , HA-Ub and Flag-tagged RIG-I. (M and N) IFN-alpha (M) and IFN-beta (N) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI=1) at 24h post-infection, and then individually knock down RIG-I or TRIM25, analyzed by qRT-PCR (n=3; means ± SEM; *p
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    AVAN direct binds to TRIM25 and enhances the antivirus immune response (A) RNA pull-down of AVAN -associated proteins using biotinylated AVAN or antisense probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. (B) Pull-down western blot showing that AVAN can bind directly to TRIM25. (C) ChIRP followed by western blot show that AVAN can bind to TRIM25. (D and E) Exogenous (D) and endogenous (E) RIP of TRIM25 in BJ501 infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR. (F) AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD or Flag-Ring. (G) Truncated AVAN pull-down, truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pulldown. (H and I) TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN s or siRNAs, followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. (J and K) Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVAN s or siRNAs. Anti-RIG-I antibody was used for immunoprecipitated. (L) Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN , HA-Ub and Flag-tagged RIG-I. (M and N) IFN-alpha (M) and IFN-beta (N) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI=1) at 24h post-infection, and then individually knock down RIG-I or TRIM25, analyzed by qRT-PCR (n=3; means ± SEM; *p

    Journal: bioRxiv

    Article Title: Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a

    doi: 10.1101/623132

    Figure Lengend Snippet: AVAN direct binds to TRIM25 and enhances the antivirus immune response (A) RNA pull-down of AVAN -associated proteins using biotinylated AVAN or antisense probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. (B) Pull-down western blot showing that AVAN can bind directly to TRIM25. (C) ChIRP followed by western blot show that AVAN can bind to TRIM25. (D and E) Exogenous (D) and endogenous (E) RIP of TRIM25 in BJ501 infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR. (F) AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD or Flag-Ring. (G) Truncated AVAN pull-down, truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pulldown. (H and I) TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN s or siRNAs, followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. (J and K) Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVAN s or siRNAs. Anti-RIG-I antibody was used for immunoprecipitated. (L) Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN , HA-Ub and Flag-tagged RIG-I. (M and N) IFN-alpha (M) and IFN-beta (N) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI=1) at 24h post-infection, and then individually knock down RIG-I or TRIM25, analyzed by qRT-PCR (n=3; means ± SEM; *p

    Article Snippet: In vitro biotin-labeled RNAs (lncAVAN and its antisense RNA) were transcribed with biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Promega) and purified using the RNeasy Mini Kit (QIAGEN).

    Techniques: Isolation, SDS Page, Silver Staining, Western Blot, Infection, Quantitative RT-PCR, Transfection, In Vitro, Incubation, Immunoprecipitation, Expressing

    Association of AFAP1-AS1 and Polycomb Repressive Complex 2. (A) RIP enrichment was determined as RNA associated with EZH2 IP relative to an input control. (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate (IP). (C) Biotinylated AFAP1-AS1 was incubated with nuclear extracts (SW480 and HCT116 cells), targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and AFAP1-AS1 (n = 3). (D, E) RNAs corresponding to different fragments of AFAP1-AS1 were treated as in (C), and associated EZH2 was detected by western blotting (n = 3). Error bars ± SD. *, P

    Journal: American Journal of Cancer Research

    Article Title: Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer

    doi:

    Figure Lengend Snippet: Association of AFAP1-AS1 and Polycomb Repressive Complex 2. (A) RIP enrichment was determined as RNA associated with EZH2 IP relative to an input control. (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate (IP). (C) Biotinylated AFAP1-AS1 was incubated with nuclear extracts (SW480 and HCT116 cells), targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and AFAP1-AS1 (n = 3). (D, E) RNAs corresponding to different fragments of AFAP1-AS1 were treated as in (C), and associated EZH2 was detected by western blotting (n = 3). Error bars ± SD. *, P

    Article Snippet: Briefly, biotin-labeled RNAs were in vitro transcribed with the Biotin RNA Labeling Mix (RocheDiagnostics, Indianapolis, IN) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA).

    Techniques: Incubation, Western Blot

    DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P

    Journal: Molecular Cancer

    Article Title: LncRNA DLEU1 contributes to colorectal cancer progression via activation of KPNA3

    doi: 10.1186/s12943-018-0873-2

    Figure Lengend Snippet: DLEU1 interacts with SMARCA1 in CRC cells. a The expression of DLEU1 in cytoplasm and nucleus of HCT8 cells was measured by qRT-PCR. U6 serves as a nuclear control. GAPDH serves as a cytoplasmic control. b SMARCA1 was a potential interactive candidate of DLEU1. Biotin-labeled DLEU1 and intron control were incubated with HCT8 cell lysates, and the enriched products were eluted and separated by SDS-PAGE electrophoresis and silver staining. The differential band appearing in DLEU1 lane was analyzed by mass spectrum. c DLEU1 associated with SMARCA1 as shown by RNA pulldown and Western blot. Biotin-labeled DLEU1 and intron control were added into HCT8 cell lysates, and pulldown assays were performed. d DLEU1 was enriched by SMARCA1 in HCT8 and SW480 cell lysates. e SMARCA1 enriched DLEU1 in HCT8 cell lysates. SMARCA1 antibody was added into cell lysates and enriched RNAs were isolated. Then enriched DLEU1 was analyzed by PCR. f DLEU1 co-localized with SMARCA1 in HCT8 cells as shown by RNA FISH. Green, DLEU1; Red, SMARCA1; Blue, DAPI. Scale bar, 10 μm. g the region of nt 1~ 400 in DLEU1 was important for the interaction with SMARCA1. h DLEU1 (nt 1~ 400) associated with SMARCA1 directly as shown by RNA EMSA assays. i The region of nt 700~ 1050 is indispensable for the function of DLEU1 in colorectal cancer. Overexpression of DLEU1 with deletion of nt 1~ 400 cannot promoted proliferation and metastasis in CC. *** P

    Article Snippet: Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix (Roche Diagnostics) and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA).

    Techniques: Expressing, Quantitative RT-PCR, Labeling, Incubation, SDS Page, Electrophoresis, Silver Staining, Western Blot, Isolation, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Over Expression

    Summary of the known interactions of the male-specific lethal (MSL) complex subunits . MSL1 and MSL2 form a DNA-binding module, the specificity of which requires the association of MSL2 with roX RNA. The association of MSL1 and MSL3 with MOF enhances its histone acetyltransferase activity and specificity. MLE and MSL2 interact and contribute to MOF activity. Maleless (MLE) is responsible for the incorporation of roX RNA in the complex and requires roX RNA to associate with the complex. The roX RNA is needed for full histone acetylation. Please see the discussion section for details of these associations.

    Journal: Epigenetics & Chromatin

    Article Title: Role of the ATPase/helicase maleless (MLE) in the assembly, targeting, spreading and function of the male-specific lethal (MSL) complex of Drosophila

    doi: 10.1186/1756-8935-4-6

    Figure Lengend Snippet: Summary of the known interactions of the male-specific lethal (MSL) complex subunits . MSL1 and MSL2 form a DNA-binding module, the specificity of which requires the association of MSL2 with roX RNA. The association of MSL1 and MSL3 with MOF enhances its histone acetyltransferase activity and specificity. MLE and MSL2 interact and contribute to MOF activity. Maleless (MLE) is responsible for the incorporation of roX RNA in the complex and requires roX RNA to associate with the complex. The roX RNA is needed for full histone acetylation. Please see the discussion section for details of these associations.

    Article Snippet: Briefly, single-stranded antisense roX1 and roX 2 probes were labeled by in vitro transcription with a biotin RNA labeling mix (RocheApplied Science, Indianapolis, IN, USA) with 1.6 kb roX1 (nucleotides 1536 to 3110) or 0.8 kb roX2 cDNA fragments in pTopo 2.1 vector as the template.

    Techniques: Binding Assay, Activity Assay