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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Clin Cancer Res. 2019 Mar 27;25(13):4038–4048. doi: 10.1158/1078-0432.CCR-18-3776

Figure 3: RNA-seq confirmation of c-MYC downregulation and target genes at 3 and 24 hours in AR-positive prostate cancer cells.

Figure 3:

RNA-seq was performed on VCaP and 22Rv1 cells treated with 10nM dBET-2 for 3 and 24 hours. Volcano plots showing the differentially expressed transcripts in both VCaP and 22rv1 cells at 3 (A) and 24 (B) hours of 10nM dBET-2 treatment plotted as a function of average fold change verses log p-value. The most downregulated transcript in both cell lines at 3 hours was MYC, serving as a positive control. There is a significant correlation between genes differentially regulated in VCaP and 22Rv1 cells treated with dBET-2. Correlations between the two cell lines are shown as Log Fold change of 22rv1 cells plotted against log fold change of VCaP cells of each of the differentially expressed transcripts at 3 (C) and 24 (D) hours of 10nM dBET-2 treatment. E) RNA-seq on Du145 cells treated with 10nM dBET-2 for 3 hours plotted as a function of average fold change verses log p-value. No expressed transcripts met significance for differential expression. Representative graph from duplicate experiments showing similar results. F) RNA-seq on DU145 cells treated with 10nM dBET-3 for 3 hours plotted as a function of average fold change verses log p-value. No expressed transcripts met significance for differential expression, similar to what was observed with dBET-2 treatment. Representative graph from duplicate experiments showing similar results. G) Western Blot confirmation of BRD4 degradation in whole cell lysate in DU145 cells treated with two independent BET-degraders, dBET-3 and ARV-825, as well as respective DMSO and Thalidomide controls. GAPDH serves as a loading control. H) Western Blot confirmation of BRD4 degradation in whole cell lysate in DU145 with dBET-2 (5nM and 10nM Treatment) compared to 10nM dBET-3 treatment for 3 hours. GAPDH serves as a loading control. I) RNA gene set pathway analysis from RNA-sequencing of VCaP and 22Rv1 cells treated with 10nM dBET-2 degrader and thalidomide control. SA Biosciences pathway level 1 Molecular Signatures Database (MSigDB) pathway analysis in VCaP and 22rv1 cells at 3 and 24 hours of 10nM dBET-2 treatment. Z-score of significance of individual pathways shown plotted between 22Rv1 (y-axis) and VCaP (x-axis).