ab4081  (Abcam)

Journal: Oncology Reports

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

doi: 10.3892/or.2014.3309

Figure Lengend Snippet: UCA1 binds to BRG1 in vitro and in vivo . (A) Electrophoresis of proteins bound to biotin-labeled sense and antisense UCA1 in NuPAGE 4–12% Bis-Tris gel. The biotin-labeled sense and antisense UCA1 were transcribed in vitro and incubated with HeLa cell lysate. The arrow shows the protein band for BRG1. (B) Western blot analysis of biotin-labeled sense and antisense UCA1-bound proteins using antibody against BRG1. (C) UCA1 binding BRG1 in 5637 cells was detected by RNA-binding protein immunoprecipitation assay. Antibody against BRG1 and mouse IgG (as a negative control) were used to pull-down RNAs in 5637 cells. The level of UCA1 was determined using real-time PCR. Data were normalized to input and are expressed as the means ± SD of three independent experiments * P<0.05 (t-test). (D) Expression profile analysis of three human bladder cancer cell lines. (Top) Total RNA was isolated and the level of UCA1 was determined using real-time PCR. GAPDH was used as an internal control. (Bottom) BRG1 was determined by western blotting. β-actin was used as an internal control. UCA1, urothelial carcinoma associated 1.

Article Snippet: Antibodies used for immunoblotting were anti-β-actin antibody (PM053; MBL, Japan) (1:5,000), anti-human p21 (3733-1; Abcam Epitomics, Cambridge, UK) (1:2,000), anti-H3K9me3 (49–1008; Novex, Carlsbad, CA, USA) (1:1,000), anti-H3K4m3 (ab8580) (1:2,000) and anti-human BRG1 antibodies (ab4081) (both from Abcam) (1:2,000).

Techniques: In Vitro, In Vivo, Electrophoresis, Labeling, Incubation, Western Blot, Binding Assay, RNA Binding Assay, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Expressing, Isolation, Control

Journal: Oncology Reports

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

doi: 10.3892/or.2014.3309

Figure Lengend Snippet: BRG1 plays a tumor suppressor role. (A) Colony formation of 5637-NC and 5637-iBRG1 cells. The cells were cultured for 14 days. Colonies were stained using crystal violet. (B) Colony formation of 5637-pcDNA3.1 and 5637-BRG1 cells. The cells were cultured for 14 days. Colonies were stained using crystal violet. (C) Growth curves of 5637 cells after transfection with BRG1 RNAi. Cellular proliferation was measured using MTT assay at 24, 48, 72, 96 and 120 h. The differences in data of 72, 96 and 120 h were significant, * P<0.05 (t-test). (D) Growth curves of 5637 cells overexpressing BRG1. Cellular proliferation was measured using MTT assay at 24, 48, 72, 96 and 120 h. The differences in data of 72, 96 and 120 h were significant, * P<0.05 (t-test). (E) The expression level of p21 in 5637-NC and 5637-iBRG1 cells was determined by real-time PCR. Data were normalized to GAPDH and are expressed as the means ± SD of three independent experiments, * P<0.05 (t-test). Western blot analysis of BRG1 and P21 in 5637-NC and 5637-iBRG1 cells. β-actin served as the internal control. (F) The expression level of p21 in 5637-pcDNA3.1 and 5637-BRG1 cells was determined by real-time PCR. Data were normalized to GAPDH and are expressed as the means ± SD of three independent experiments, * P<0.05 (t-test). Western blot analysis of BRG1 and p21 in 5637-pcDNA3.1 and 5637-BRG1 cells. β-actin served as the internal control.

Article Snippet: Antibodies used for immunoblotting were anti-β-actin antibody (PM053; MBL, Japan) (1:5,000), anti-human p21 (3733-1; Abcam Epitomics, Cambridge, UK) (1:2,000), anti-H3K9me3 (49–1008; Novex, Carlsbad, CA, USA) (1:1,000), anti-H3K4m3 (ab8580) (1:2,000) and anti-human BRG1 antibodies (ab4081) (both from Abcam) (1:2,000).

Techniques: Cell Culture, Staining, Transfection, MTT Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Oncology Reports

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

doi: 10.3892/or.2014.3309

Figure Lengend Snippet: UCA1 antagonizes the tumor suppressing function of BRG1. (A) (Left) Colony formation of EJ control, EJ-BRG1 (overexpressed BRG1) and EJ-BRG1-UCA1 (overexpressed BRG1 and UCA1) cells. The cells were cultured with G418 for 10 days. Colonies were stained using crystal violet. (Right) Colony formation of 5637 control, 5637-iUCA1 (UCA1 knocked down) and 5637-iUCA1-iBRG1 (UCA1 and BRG1 both knocked down) cells. The cells were cultured with G418 and puromycin for 10 days. Colonies were stained using crystal violet. (B) (Left) Growth curves of EJ control, EJ-BRG1 and EJ-BRG1-UCA1 cells. Cellular proliferation was measured using MTT assays at 24, 48, 72, 96 and 120 h. The differences between EJ-BRG1 and EJ-BRG1-UCA1 were significant. * P<0.05 (t-test). (Right) Growth curves of 5637 control, 5637-iUCA1 and 5637-iUCA1-iBRG1 cells. Cellular proliferation was measured using MTT assays at 24, 48, 72, 96 and 120 h. The differences between 5637-iUCA1 and 5637-iUCA1-iBRG1, 5637-iUCA1 and control were significant * P<0.05 (t-test). (C) (Left) The expression levels of UCA1 in EJ cells control EJ-BRG1 and EJ-BRG1-UCA1 were determined by real-time PCR. Data were normalized to GAPDH and are expressed as the means ± SD of three independent experiments, the UCA1 level in EJ-BRG1-UCA1 was significantly high, * P<0.05 (t-test). The expression levels of BRG1 and p21 were determined by western blotting with β-actin as the internal control. (Right) The expression levels of UCA1 in 5637 cells control 5637-iUCA1 and 5637-iUCA1-iBRG1 were determined by real-time PCR. Data were normalized to GAPDH and are expressed as the means ± SD of three independent experiments. The effect of RNAi was significant * P<0.05 (t-test). The expression levels of BRG1 and p21 were determined by western blotting with β-actin as the internal control. UCA1, urothelial carcinoma associated 1.

Article Snippet: Antibodies used for immunoblotting were anti-β-actin antibody (PM053; MBL, Japan) (1:5,000), anti-human p21 (3733-1; Abcam Epitomics, Cambridge, UK) (1:2,000), anti-H3K9me3 (49–1008; Novex, Carlsbad, CA, USA) (1:1,000), anti-H3K4m3 (ab8580) (1:2,000) and anti-human BRG1 antibodies (ab4081) (both from Abcam) (1:2,000).

Techniques: Control, Cell Culture, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

doi: 10.3892/or.2014.3309

Figure Lengend Snippet: UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P<0.05 (t-test). (C) Micrococcal nuclease assay of 5637-NC, 5637-iUCA1. Same amounts of DNA were digested with micrococcal nuclease and electrophoresed. The image shows that nuclease digestion produced a laddering pattern. It is evident that DNA from 5637-iUCA1 is more sensitive to nuclease digestion. (D) Western blotting to detect histone proteins H3K4m3, H3K9m3 in 5637-NC, 5637-iUCA1. (E) ChIP analysis of BRG1 binding to the p21 promoter in EJ-BRG1, EJ-BRG1-UCA1. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. Overexpression of UCA1 in EJ-BRG1 led to decreased occupancy of p21 promoter by BRG1, * P<0.05 (t-test). UCA1, urothelial carcinoma associated 1.

Article Snippet: Antibodies used for immunoblotting were anti-β-actin antibody (PM053; MBL, Japan) (1:5,000), anti-human p21 (3733-1; Abcam Epitomics, Cambridge, UK) (1:2,000), anti-H3K9me3 (49–1008; Novex, Carlsbad, CA, USA) (1:1,000), anti-H3K4m3 (ab8580) (1:2,000) and anti-human BRG1 antibodies (ab4081) (both from Abcam) (1:2,000).

Techniques: Activity Assay, Binding Assay, Control, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Nuclease Assay, Produced, Western Blot, Over Expression

Journal: Oncology Reports

Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

doi: 10.3892/or.2014.3309

Figure Lengend Snippet: UCA1 expression correlates positively with BRG1 expression in bladder cancer tissue samples. (A) Real-time PCR was used to analyze UCA1 and BRG1 mRNA levels in the same samples. S, sample. (B) The status of UCA1 and BRG1 expression in bladder cancer specimens, >2-fold was identified as high expression. The 2×2 correlation table and Fisher’s exact test were used. The two-sided value P=0.009 was considered significant. UCA1, urothelial carcinoma associated 1.

Article Snippet: Antibodies used for immunoblotting were anti-β-actin antibody (PM053; MBL, Japan) (1:5,000), anti-human p21 (3733-1; Abcam Epitomics, Cambridge, UK) (1:2,000), anti-H3K9me3 (49–1008; Novex, Carlsbad, CA, USA) (1:1,000), anti-H3K4m3 (ab8580) (1:2,000) and anti-human BRG1 antibodies (ab4081) (both from Abcam) (1:2,000).

Techniques: Expressing, Real-time Polymerase Chain Reaction