Journal: EMBO Reports

Article Title: BRD4 isoforms have distinct roles in tumour progression and metastasis in rhabdomyosarcoma

doi: 10.1038/s44319-023-00033-1

Figure Lengend Snippet: ( A ) MSTN protein level was analysed in shScr, shBRD4-L and shBRD4-S cells. β-Actin was used as loading control. Images are representative of at least three biological replicates. Densitometric analysis was done to quantify MSTN expression relative to β-Actin from three independent experiments and is shown in the bar graph. Error bars correspond to the average ± SEM ( n = 3 biological replicates). Two-tailed non-parametric unpaired t test was performed for statistical analysis. *** p ≤ 0.001. ( B ) Tumour lysates from 2 independent RD shScr, shBRD4-L and shBRD4-S xenografts were analysed for BRD4, MHC and MSTN expression using Western blotting. β-Actin was used as loading control. ( C – E ) ChIP assay was performed to examine the enrichment of BRD4-L and BRD4-S isoforms ( C ), H3K9Ac ( D ) and RNA Pol II ( E ) on the MSTN promoter relative to IgG control. The values were plotted as percentage of input, the average ± SEM is shown (n = 4 biological replicates). Two-tailed non-parametric unpaired t test was performed for statistical analysis. **** p ≤ 0.0001. ( F , G ) ChIP assay to examine the enrichment of BRD4-L ( F ) and RNA Pol II ( G ) in shBRD4-L and shBRD4-S compared to shScr control cells on MSTN promoter. The values were plotted as percentage of input, average ± SEM ( n = 4 biological replicates). Two-tailed non-parametric unpaired t test was performed for statistical analysis. **** p ≤ 0.0001, n.s. not significant. ( H ) BrdU assay in shScr, shBRD4-L and shBRD4-S cells and treated with or without 1 μg/ml of recombinant myostatin protein for 72 h. Nuclei were stained with DAPI (blue). Images are representative of at least three biological replicates. Scale bar: 100 μm. Scatter plot showing the percentage of BrdU + cells in shBRD4-L, shBRD4-S in comparison with shScr cells treated with or without recombinant myostatin protein. The values correspond to the average ± SEM ( n = 3 biological replicates with 3 technical replicates shown). Statistical significance was calculated by one-way ANOVA analysis. **** p ≤ 0.0001. ( I ) shScr, shBRD4-L and shBRD4-S cells were cultured in differentiation media in the absence or presence of 1 μg/ml of recombinant myostatin protein for 72 h. MHC + cells were analysed by immunofluorescence with anti-MHC antibody. Images are representative of at least three biological replicates. Scale bar: 100 μm. Scatter plot indicating MHC + cells in shBRD4-L, shBRD4-S in comparison with shScr cells with or without recombinant myostatin protein. The values correspond to the average ± SEM ( n = 3 biological replicates with 3 technical replicates shown). Statistical significance was calculated by one-way ANOVA analysis. **** p ≤ 0.0001. ( J ) Western blot analysis indicating protein expression of MHC and MSTN in shScr, shBRD4-L and shBRD4-S cells with or without recombinant myostatin protein for 72 h. β-Actin was used as loading control. .

Article Snippet: ChIP was conducted with 2 µg of IgG or purified antibodies against BRD4-L (Cat. #: 13440S, CST, 1:50), H3K9Ac (Cat. #: ab4441, Abcam) and RNA Pol II (Ser2P) (Cat. #: ab193468, Abcam) or 1 µg of BRD4-S (Wu et al, ) and analysed by qPCR using 4% of IP products and 0.2% of input DNA.

Techniques: Expressing, Two Tailed Test, Western Blot, BrdU Staining, Recombinant, Staining, Comparison, Cell Culture, Immunofluorescence

Journal: EMBO Reports

Article Title: BRD4 isoforms have distinct roles in tumour progression and metastasis in rhabdomyosarcoma

doi: 10.1038/s44319-023-00033-1

Figure Lengend Snippet: ( A ) Western blot analysis of ITGA4 and ITGA5 in shScr, shBRD4-L and shBRD4-S cells. β-Actin was used as loading control. Images are representative of at least three biological replicates. ITGA4 and ITGA5 expression relative to β-Actin from three independent experiments was quantified densitometrically and is shown in the bar graph. Error bars correspond to the average ± SEM ( n = 3 biological replicates). Two-tailed non-parametric unpaired t test was performed for statistical analysis. *** p ≤ 0.001. ( B ) Tumour lysates from 2 independent NOD/SCID mice from tail vein assays were analysed for expression of ITGA4 and ITGA5 using Western blotting. β-Actin was used as loading control. ( C ) ChIP assay was performed to examine the enrichment of BRD4-L and BRD4-S on ITGA4 and ITGA5 promoter, IgG was used as a control. The values were plotted as percentage of input, average ± SEM ( n = 4 biological replicates). Two-tailed non-parametric unpaired t test was performed for statistical analysis. ** p ≤ 0.01, **** p ≤ 0.0001. ( D , E ) ChIP assay was performed to examine the enrichment of H3K9Ac ( D ) and RNA Pol II ( E ) on the ITGA4 and ITGA5 promoter. The values were plotted as percentage of input, average ± SEM ( n = 4 biological replicates). Two-tailed non-parametric unpaired t test was performed for statistical analysis. *** p ≤ 0.001, n.s. = not significant. ( F – H ) Enrichment of BRD4-L ( F ), BRD4-S ( G ) and RNA Pol II ( H ) in shScr, shBRD4-L and shBRD4-S cells at the ITGA4 and ITGA5 promoter was analysed by ChIP assay The values were plotted as percentage of input, the average ± SEM ( n = 4 biological replicates) is shown. Two-tailed non-parametric unpaired t test was performed for statistical analysis. n.s. not significant, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. ( I ) Upon transient knockdown of BRD4-L in shBRD4-S cells for 48 h, migratory and invasive capacity of shScr, shBRD4-L and shBRD4-S cells was analysed using transwell assay. The inserts were stained with crystal violet. Images are representative of at least three biological replicates. Scale bar: 100 μm. Scatter plots show the percentage of migrated and invaded shScr, shBRD4-L, shBRD4-S and shBRD4-S + siBRD4-L cells after 48 h. The values correspond to the average ± SEM ( n = 5 biological replicates with 2 technical replicates shown). Statistical significance was calculated by one-way ANOVA analysis. **** p ≤ 0.0001. ( J ) Western blot analysis of ITGA4 and ITGA5 in shScr, shBRD4-L, shBRD4-S and shBRD4-S + siBRD4-L. β-Actin was used as loading control. .

Article Snippet: ChIP was conducted with 2 µg of IgG or purified antibodies against BRD4-L (Cat. #: 13440S, CST, 1:50), H3K9Ac (Cat. #: ab4441, Abcam) and RNA Pol II (Ser2P) (Cat. #: ab193468, Abcam) or 1 µg of BRD4-S (Wu et al, ) and analysed by qPCR using 4% of IP products and 0.2% of input DNA.

Techniques: Western Blot, Expressing, Two Tailed Test, Transwell Assay, Staining