Fig. 3: BET inhibitors potentiate activation of p53 target genes by p53.
From: BRD4-mediated repression of p53 is a target for combination therapy in AML
a Western blot of C-MYC and p53 in OCI-AML3 cells treated for 24 h with vehicle, 200 nM CPI203, 2.5 μM nutlin-3, and the drug combination. b Western blot for BCL-2 in OCI-AML3 cells treated for 24 h with vehicle, 200 nM CPI203, 2.5 μM nutlin-3, and the drug combination. c Western blot for BCL-2 comparing the control OCI-AML3 and cells over-expressing BCL-2. d Cell killing as assessed by flow cytometry using Annexin-V and PI staining of OCI-AML3 control (empty vector) cells versus OCI-AML3 over-expressing BCL-2, according to treatment condition (p-value >0.05 by two-tailed unpaired t-test, Means ± SD are shown, n = 3). e A heat map of genes synergistically regulated by the drug combination determined by RNA-seq of OCI-AML3 cells according to treatment condition (24 h). Genes synergistically up-regulated (Up) by the drug combination were rigorously identified as those where C/(A + B) = > 1.25 and combination FPKM > DMSO FPKM, and synergistically down-regulated genes (Down) where C/(A + B) = > 1.25 and combination FPKM < DMSO FPKM (where C = combination FPKM – DMSO FPKM; A = nutlin-3 FPKM – DMSO FPKM; B = CPI203 FPKM – DMSO FPKM). f A heat map of 24 high confidence p53 target genes that are synergistically up-regulated or down-regulated in OCI-AML3 according to treatment condition. g qPCR analysis of CDKN1A expression in OCI-AML3 under indicated treatments for 24 h (**=p ≤ 0.01, two-tailed unpaired t-test, Means ± SD are shown, n = 3). h qPCR analysis of BBC3 expression in OCI-AML3 under indicated treatments for 24 h (****=p ≤ 0.0001, two-tailed unpaired t-test, Means ± SD are shown, n = 3). i Western blot analysis of p53 targets BBC3 and CDKN1A in OCI-AML3 according to treatment condition. j qPCR expression analysis of TP53INP1 in OCI-AML3 under indicated treatments for 24 h (*=p ≤ 0.05, two-tailed unpaired t-test, Means ± SD are shown, n = 3). k qPCR expression analysis of GDF15 in OCI-AML3 under indicated treatments for 24 h (**=p ≤ 0.01, two-tailed unpaired t-test, Means ± SD are shown, n = 3). l qPCR expression analysis of FUCA1 in OCI-AML3 under indicated treatments for 24h (*=p ≤ 0.05, two-tailed unpaired t-test, Means ± SD are shown, n = 3). m Western blot analysis of p53 and CDKN1A in OCI-AML3 cells and OCI-AML3 cells harboring shRNA p53 according to treatment condition. n A heat map of an RNA-seq data of 19 high confidence p53 target genes that are synergistically up-regulated by the drug combination in control (empty-vector) OCI-AML3 cells, versus shRNA p53 OCI-AML3 cells, according to treatment condition. o Western blot analysis of BBC3 and NOXA in control (empty vector) OCI-AML3 cells, and OCI-AML3 cells harboring CAS9 and sgRNAs against PUMA and NOXA. p FACS analysis of cell death (annexin VI and PI) in control OCI-AML3 cells or cells in which PUMA and NOXA were knocked out by CRISPR/CAS9, under indicated conditions for 72 h (*=<additive. **=>additive, two-tailed unpaired t-test, Means ± SD are shown, n = 3).
