Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of Pol II transcription cycle in normal (N) tissue and tumor (T) of the large intestine. RNA expression level of Pol II transcription cycle-associated genes in GTEx and TCGA databases were analyzed. I initiation, E elongation, T termination. ( B ) Volcano plot for fold change of T vs N of 104 Pol II transcription-associated genes in COAD. NELFCD and SUPT4H1 genes are indicated with an arrow. NELFCD gene is highlighted in red. Statistical test: differential expression analysis was performed using DESeq2 (negative binomial generalized linear model with Wald test); P values were adjusted for multiple testing using the Benjamini–Hochberg method (adj.P.Val). The numbers of tumor (T) and normal (N) samples for COAD are provided in Table . ( C ) Western blot of NELF-C-AID DLD-1 whole-cell extract (WCE) using the indicated antibodies. The treatment time (h) of IAA is also indicated. Western blot image of NELF-C is reused in ( G ). ( D ) Western blot of SPT4-AID DLD-1 WCE using the indicated antibodies. The treatment time (h) of IAA is also indicated. ( E ) Western blot of NELF-C-AID DLD-1 cell nuclear fraction using the indicated antibodies. The treatment time (h) of IAA is indicated. Western blot images of NELF-C and Pol I are reused in Fig. . ( F ) Cell count ratios of 12, 24, and 36 h IAA to 0 h IAA are shown. Parental, NELF-C-AID, and SPT4-AID DLD-1 cells are displayed in light gray, red, and dark gray, respectively. Error bars represented the mean ± SEM (biological replicates, n = 4). Statistical test: paired two-sample t test (Student’s t test), two-sided, performed within each Group comparing the same Dish across adjacent timepoints (0 h vs 12 h, 12 h vs 24 h, 24 h vs 36 h) on the 0h-normalized ratios. P values are shown. n.s. (not significant, P > 0.05). ( G ) Western blot of NELF-C-AID DLD-1 whole-cell extract (WCE) using the indicated antibodies. The treatment time (h) of IAA is indicated. Western blot image of NELF-C is reused in ( C ). ( H ) Cell cycle (%) of parental and NELF-C-AID DLD-1 cells following the indicated treatment time (h) of IAA. Error bars represented the mean ± SEM (biological replicates, n = 3). .

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: RNA Expression, Quantitative Proteomics, Western Blot, Cell Characterization

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Log 2 fold change of T vs N of the Pol II transcription-associated genes. Upregulated (>1) and down-regulated (<-1) genes are highlighted in red and blue, respectively. NELFCD gene is indicated with an arrow. ( B ) Volcano plot of the log2 fold change of Tumors (T) vs Normal tissues (N) on 835 cell cycle-related genes in COAD. CDKN1A , CDKN1B , and CDKN1C genes are indicated in red. The numbers of tumor (T) and normal (N) samples for COAD are provided in Table . Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. ( C ) Log2 of the RNA expression levels of NELFCD , SUPT4H1 , CDKN1A , and CDKN1C genes in N and T of the indicated tissues and tumors are compared. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Statistical test: Wilcoxon signed-rank test. P values are shown. The numbers of tumor (T) and normal (N) samples for each tissue types are provided in Table . ( D ) Log 2 fold change of T vs N of NELFCD across indicated tissue types. ( E ) Comparative proteomic analysis of NELF subunits and SPT4 in human primary colon cancers and its adjacent tissues. Statistical test: negative binomial distribution-based Wald test. P values are shown. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( F ) Schematic model of transcription addicted by NELF in tumor.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: RNA Expression

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of POINT-seq strategy. Chromatin of NELF-C-AID or SPT4-AID DLD-1 cells was stringently isolated using Urea and Empigen detergent. Pol II intact nascent transcript (POINT) was precipitated with Pol II CTD antibody from DNA-digested chromatin fraction. ( B ) Example view of POINT-seq on RPS23 gene in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. ( C ) Metagene analysis of POINT-seq on scaled transcription unit −/+2.5 kb in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. ( D ) Violin plots of Termination Index (TI) in the indicated cell lines treated for 4 or 3 h with DMSO or IAA. Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median. ( E ) Example view of SIRV-normalized POINT-seq on RPS23 and HELLS genes in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). ( F ) Metagene analysis of POINT-seq on scaled transcription unit −/+2.5 kb in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). ( G ) Violin plots of TI in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. ** P = 0.0021, **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Isolation

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Example view of TT-seq on PTPN13 gene in the indicated cell line treated for 4 h with DMSO or IAA, followed by NVP-2 treatment for 0, 0.5, or 1 h. ( B ) Profiles of TT-seq mean signal in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA, followed by NVP-2 treatment for 0, 0.5, or 1 h. Data for 2 biological replicates with ≥300 kb genes are shown. ( C ) Box plots of elongation activity (EA) derived from TT-seq. Data for short and long genes are shown. n = 65. Statistical test: Wilcoxon rank-sum test. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). P value is 0.029. ( D ) Scatter plot showing the correlation between EA and TI. The regression line (red) and the 95% confidence interval (green) are also shown. n = 129. Statistical method: Spearman correlation. P value is 0.036. ( E ) Metagene of POINT-seq, TT-seq, Pol II EI (TT-seq/POINT-seq) for non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( F ) Box plots of Pol II EI (TT-seq/POINT-seq and TT-seq/PRO-seq) for non-overlapping pc genes (n = 3459) in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Statistical test: Kruskal–Wallis test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( G ) Pol II EI for indicated three gene-length classes of normalized non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( H ) INTS3 ChIP-seq profile across normalized transcription units of non-overlapping pc genes in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. ( I ) Box plots of the log2 of the Dox/Ctrl termination index, two biological replicates in all genes, genes with transcription readthrough which is based on previous study (Dasilva et al, ), and NELF-C KD affected genes (top 25%, highlighted in orange box). 0: no change in TI index upon shCtrl or shINTS11 induction by Dox. The number of genes in each category is indicated on the figure. Statistical test: Kruskal–Wallis test. * P = 0.039, ** P = 0.0019, **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%).

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Activity Assay, Derivative Assay, ChIP-sequencing

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Schematic diagram of Pol II elongation index (EI) analysis. Pol II EI is determined as TT/POINT. 4sU (Green circle), Pol II (blue oval). ( B ) Pol II EI for all expressed pc genes across normalized transcription units in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Last exon (LE)-0.5 kb ~ +2.5 kb window is highlighted in gray. ( C ) Pol II EI for non-overlapping pc genes across LE. ( D ) Metagene of CSTF64 and XRN2 ChIP-seq for non-overlapping pc genes across normalized transcription units (top) and LE (bottom) in NELF-C-AID DLD-1 cells treated for 4 h with DMSO or IAA. Last exon (LE)-0.5 kb ~ +2.5 kb window is highlighted in gray. ( E ) Quantification of CSTF64 and XRN2 ChIP-seq signals (IAA/DMSO) in 2.5 kb DoG regions of non-overlapping pc genes. Statistical test: Kruskal–Wallis test. **** P < 0.0001. Violin plot: minimal-to-maximal value, box center line: median.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: ChIP-sequencing

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Example view of SIRV-normalized POINT-seq on CD58 gene adjacent to a RI zone in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). DNA replication initiation (RI) zone is highlighted in gray. RI score: 54.6579. Re-analyzed Pu-seq data shows RI zones in green. ( B ) Metagene analysis of POINT-seq on scaled RI zones −/+2.5 kb in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). ( C ) Box plots of SIRV-normalized POINT-seq signals in RI zones in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Two biological replicates are shown. Statistical test: Wilcoxon signed-rank test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( D ) Scatter plots of SIRV-normalized POINT-seq signals and RI score in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). Cut-off value is Log 2 (1.25) in SIRV-normalized POINT-seq. Pol II-affected (P-A) and not affected (P-NA) zones are classified as higher and lower than the cut-off value, respectively. RI zones that are above the cut-off in the three timepoints (4, 12, 24 h IAA) are indicated in red. ( E ) Example view of SIRV-normalized POINT-seq on CD2AP gene adjacent to a P-NA zone in NELF-C-AID DLD-1 cells (4 h DMSO and IAA). DNA RI zone is highlighted in gray. RI score: 42.8401. ( F ) Box plots of SIRV-normalized POINT-seq signals in P-A ( n = 5878) and P-NA ( n = 6651) RI zones in NELF-C-AID DLD-1 cells (0, 4, 12, and 24 h IAA). Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( G ) Size in bp of P-A ( n = 5878) or P-NA ( n = 6651) RI zones. Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%). ( H ) Distance in bp between the closest gene and the P-A ( n = 5878) and P-NA ( n = 6651) RI zones. Statistical test: Wilcoxon rank-sum test. **** P < 0.0001. Box plot: minimal-to-maximal value, box center line: median, bounds of box: interquartile (25 and 75%).

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques:

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Representative images of PLA with the indicated antibodies after 4 h DMSO and IAA in NELF-C-AID DLD-1 cells. Blue: Hoechst, Red: PLA. Scale bar size is 5 µm. ( B ) Box plots of the PLA foci per nucleus after 4 h DMSO and IAA in NELF-C-AID DLD-1 cells. The PLA was performed with indicated antibodies. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Dots represent individual nuclei. Statistical test: Brunner–Munzel test. Statistical test: Wilcoxon rank-sum test. P values are shown. not significant (n.s.). ( C ) Western blot of chromatin fraction of NELF-C-AID DLD-1 cells (0, 4, and 24 h IAA) using the indicated antibodies. ( D ) Cell cycle (%) of 24 h DMSO or MLN-4924 treated NELF-C-AID DLD-1 cells. The cells were pre-treated with IAA or DMSO for 24 h. Error bars represented the mean ± SEM (biological replicates, n = 3). ( E ) BrdU dot blot assay to evaluate global DNA synthesis. ( F ) BrdU-IP-seq of two biological replicates (R1 and R2) for FOCAD gene regions in parental and NELF-C-AID DLD-1 cells treated with IAA for 4 h. IP efficiencies (IP/input) are shown. BrdU plus (magenta) and minus (green) zones in Parental minus NELF-C-AID (Δ). Normalized POINT-seq profiles for (+) and (−) strands in NELF-C-AID DLD-1 cells treated with IAA for 0 h (DMSO) or 4 h (IAA) are shown. Pol II transcription termination defect are indicated by arrows. FOCAD DoG region is highlighted by gray. Early and Late S phase are separated by dashed line. ( G ) BrdU-IP-qPCR analysis to assess local DNA synthesis rates in region exhibiting termination defect. The top panel shows the locations of primer sets used for qPCR. The bottom panel displays the IP/input values for each locus, normalized to the ELAVL2 locus, a late-replicating region that is not expected to replicate at the onset of the S phase (ELAVL2 locus = 1). Error bars represented the mean ± SEM (biological replicates, n = 3). P values are indicated.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Western Blot, Dot Blot, DNA Synthesis

Journal: EMBO Reports

Article Title: NELF prevents transcriptional readthrough into DNA replication zones in cancer cells

doi: 10.1038/s44319-026-00700-z

Figure Lengend Snippet: ( A ) Representative images of PLA with the indicated antibodies after 4 h DMSO and IAA in MCM2 (right) or PCNA (left) expressing NELF-C-AID DLD-1 cells. Blue: Hoechst, Green: MCM2 or PCNA, Red: PLA. Scale bar size is 5 µm. ( B ) Box plots of the PLA foci per nucleus after 4 h DMSO and IAA in MCM2 or PCNA expressing NELF-C-AID DLD-1 cells. The PLA was performed with the indicated antibodies. Box plots show the median (center line) and interquartile range (box, 25th–75th percentiles); whiskers indicate 1.5×IQR. Dots represent individual nuclei. Statistical test: Brunner–Munzel test. P values are shown. n.s. (not significant). ( C ) Schematic representation of the BrdU-IP assay performed following release from M phase. ( D ) BrdU-IP-seq of two biological replicates (R1 and R2) for the indicated chromosome 3 window in parental and NELF-C-AID DLD-1 cells treated with IAA for 4 h. IP efficiencies (IP/input) are shown. BrdU plus (magenta) and minus (green) zones in Parental minus NELF-C-AID (Δ). Dashed lines indicate 1.25 in BrdU-IP (%). Normalized POINT-seq profiles for (+) and (−) strands in NELF-C-AID DLD-1 cells treated with IAA for 0 h (DMSO) or 4 h (IAA) are shown. Pol II transcription termination defect are indicated by arrows. ( E ) Quantification of the POINT-seq (IAA 4 h – DMSO) signal over minus and plus BrdU zones located downstream of expressed pc genes (from TES + 2.5 to TES + 5 kb). Violin plots show the distribution (kernel density) of log10 POINT-seq read coverage mean values. Embedded box plots indicate the median (center; white dot/line) and the interquartile range (box; 25th–75th percentiles). Whiskers extend to 1.5×IQR; whisker endpoints represent the minimum and maximum values within this range. Statistical test: Brunner–Munzel test. **** P < 0.0001. ( F ) Model of NELF-mediated transcription termination and DNA replication initiation or elongation. With NELF, Pol II transcription is terminated at proximal PAS before Pol II reaches DNA RI zone. Without NELF, Pol II transcription is extended to distal PAS and impair replication in the RI zone. This perturbs DNA RI and/or causes T-R conflict (HO, Head-ON), resulting in cell quiescence.

Article Snippet: Next, primary antibodies were diluted as follows: PCNA (SC-56, Santa Cruz) and MCM2 (12265, Cell Signalling) at 1:500, and Pol II Ser2P (NB100-1805, Novus) at 1:1000.

Techniques: Expressing, Whisker Assay

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