Fig 1: Baseline and post-OTX015 expression of BRD2/3/4 and c-MYCBasel expression of BRD2/3/4 and c-MYC in terms of protein (A) and mRNA (B) levels were evaluated in the three cell lines by Western blotting and qRT-PCR, respectively. Protein (C) and mRNA (D) levels were evaluated after 24-, 48- and 72-h OTX015 (650 nM in HCC1937 and MDA-MB-468 cells, and 75 nM in the MDA-MB-231 cell line). Significant differences in mRNA levels were determined by one-way ANOVA test (p < 0.01) followed by an SNK a posteriori test (*p < 0.05, **p < 0.01). Western blots are representative of at least three independent experiments. Bars and vertical lines represent the mean ± SEM, respectively (n = 3).
Fig 2: Design and development of a heterozygous knock-in BromoTag-Brd2 HEK293 cell line. (A) Design of the knock-in construct used in the development of the CRISPR construct. (B) FACS single cell sort of HEK293 cells based on GFP expression. Successive single cells were sorted into individual wells of a 96-well plate. (C) Junction PCR using genomic DNA of an expanded GFP-expressing clone paired against parental HEK293. (D) Western blot demonstrating the selectivity of the polyclonal Brd4BD2L387A. antibody.
Fig 3: BRD2, 3, and 4 Are Replication Fork Proteins Required for Fork Progression(A) U2OS cells were transfected with non-targeting (siNT) or BRD3 siRNAs, treated with increasing concentrations of ATR inhibitor (VX-970) and measured for viability after 72 h. Mean ± SD, n = 3, two-way ANOVA with Dunnett post-test.(B) Immunoblot analysis to verify siRNA knockdown.(C–F) Fork speeds were measured in (C, E, and F) U2OS or (D) RPE-hTERT cells transfected with siRNAs and labeled with IdU and CldU as indicated. Mean, ANOVA with Dunnett post-test (C, D, and F) or two-tailed t test (E). In (F) cells were pre-incubated with DRB for 1 h prior to labeling and maintained during labeling. siRNA pools were used in (D) and (E).See also Figure S4.
Fig 4: BRD4 mediates the profibrotic effect of BETs in dcSSc fibroblasts.(A) BRD2 knockdown in dcSSc fibroblasts led to upregulation of COL1A1, and BRD3 knockdown resulted in downregulation of TIMP3. Knockdown of BRD4 resulted in downregulation of COL1A1, ACTA2, TGFB, CTGF, and MMP1. n = 5–6 patients. (B) BRD4 knockdown significantly inhibited gel contraction in dcSSc fibroblasts, whereas BRD2 or BRD3 knockdown had minimal effect on gel contraction. n = 4–5 patients. Results are expressed as mean ± SD and P < 0.05 was considered significant. (A and B) Significance was determined by unpaired 2-tailed t test or Mann-Whitney U test.
Fig 5: Biological evaluation of second-generation B&H–PROTACs in BromoTag-Brd2 HEK293 cells. Western blot data for BET protein levels monitored from 10 µM to 1 nM compound treatment over 6 h in heterozygous BromoTag-Brd2 HEK293 cells. Bands are normalized to tubulin and negative control (cis-MZ1) to derive DC50 values that enable the rank order of each PROTAC.
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