Figure 1.

NSC59984 treatment re-activates WT-p53 signaling and intrinsic apoptosis. (A) Panel of EAC cells studied, providing their biological origin and associated TP53 status. (B) Cellular proliferation was determined by fold change in CyQUANT measurement of cellular DNA following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. A 2-way ANOVA test with Tukey correction was carried out for statistical analysis. (C) Analysis of apoptosis in EAC cells as measured by change in percentage of Annexin V positive cells following treatment with NSC59984 (12 μM) for 72 hr. A 2-way ANOVA test with Tukey correction was carried out for statistical analysis. (D) Whole cell lysate from EAC cells was blotted for p53 and p73 expression following 8 hr treatment with NSC59984 (12 μM). β-Actin served as the loading control. Numbers represent fold change in expression relative to changes in β-Actin. (E) Fold change in mRNA expression of PUMA analyzed by RT-qPCR following 3 hr treatment NSC59984 (6, 12 or 25 μM). Expression was normalized to β-actin, and changes in carrier-treated cells were set to 1. A 2-way ANOVA test with Dunnett’s correction was carried out for statistical analysis. (F) Whole cell lysate from EAC cells was blotted for Bax and p21 expression following 8 hr treatment with NSC59984 (6, 12 or 25 μM). β-Actin served as the loading control. (G) ChIP analysis of the occupancy of p53 on the p21 promoter in carrier-treated cells or following 2 hr treatment with 12 μM NSC59984 in CP-A-WT or ESO26-R248W cells. ChIP was performed using the Active Motif ChIP-IT Express^® kit. Chromatin Input was diluted 1:100 before analysis (left), ChIP DNA was analyzed as percent of Input (right). Raw Ct values can be found in Supplemental Table 1. A 2-way ANOVA with Šidák’s correction was carried out for statistical analysis. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.