Activation of a p53-dependent transcriptional program during erythroid differentiation. Human primary erythroblasts were cultured with SCF, IL-6, and IL-3 for 6 days (with the addition of dexamethasone at day 1) and then with SCF and EPO between days 7 and 11. CX-5461 (50 nM) was added at day 9 for 48 hours, and control or treated cells were collected at day 11. (A) Western blot showing p53 activation by CX-5461. Actin was used as the loading control. Representative results of 3 independent experiments. (B) GSEA of p53 pathway genes in erythroblasts treated by CX-5461 or vehicle. NES, normalized enrichment score. (C) GO term enrichment analysis of differentially expressed genes between treated and untreated erythroblasts with CX-5461 for 48 hours. (D) Venn diagram of the repartition of p53 direct target genes identified by ChIP-seq in CX-treated and control erythroblasts. Lost, control; shared, control or CX-5461; gained, CX-5461. (E) ChIP-seq density heat maps showing the landscape of p53 target genes in control and CX-5461 treatment conditions. (F) GO analysis of the 70 upregulated p53 direct target genes after CX-5461 treatment. (G) Volcano plot representing p53 targets identified by ChIP-seq, either shared by control and CX-5461 treatment conditions (green) or detected in control conditions (yellow), the expression of which in log2 (FC) is upregulated by CX-5461. FC, fold change. (H) Heat map visualization of the most representative p53 direct target genes upregulated after CX-5461 treatment. (I) Quantification of p53 direct target gene expression after 48 and 96 hours of CX-5461 treatment by qRT-PCR. *P < .05, **P < .01, by Student t test. (J) Combined analysis of p53 peaks in selected genes in control and CX-5461 treatment conditions and GATA1, H3K27me3, H3K27, H3K4me3, and RNA Pol II (RNAPII) peaks identified in human proEs by Xu et al³¹  and Huang et al.³²