Fig. 5

Cross-talk of p53 activation and INFγ stimulation. A, B RT-qPCR of IRF1 (A) and CD274 (B) in A375 cells treated with INFγ (100 ng/mL, horizontally striped column) or AMG-232 (1 μM, vertically striped column) or their combination (hatched column) for 24 h. mRNA levels were normalized to ACTB mRNA (n = 3 biological replicates, each with technical triplicates). Relative levels are shown as fold change ± SEM compared with untreated A375 (*P < 0.01) or cells treated only with IFNγ and its combination with AMG-232 (P < 0.01). C Western blots of A375 cells treated as in (A) and (B). PD-L1, IRF1, SOX10, p-STAT1, p53, MDM2, and β-actin (as loading control) have apparent molecular weights of 55, 55, 55, 80, 90, and 42 kDa, respectively. Stripped and re-probed blots are indicated by arrows. D Flow cytometric analysis of membrane PD-L1 in A375 cells treated as in (A) and (B). PD-L1 levels were normalized to untreated A375 cells and are presented as the mean fluorescence intensity of replicates ± SD (n = 3), *P < 0.01. E NK cell-mediated cytotoxicity assay in A375 cells treated as in (A) and (B). Co-culture was performed at 10:1 (NK: tumor cell) ratio. The percentage of dead cells (7-AAD+) in the tumor cell population (CFSE+) is shown. Data are presented as mean ± SEM (n = 3 biological replicates) for cells treated only with IFNγ compared with its combination with AMG-232 (P < 0.01). F Resazurin viability assay of A375 cells treated with only AMG-232 (black line) at the indicated concentrations, or in combination with 100 ng/ml INFγ (grey line) for 72 h. Each sample was measured in five replicates. G NK cell-mediated cytotoxicity in A375 (white columns) and A375p53KO (grey columns) at the indicated NK: tumor cell ratios. The percentage of dead tumor cells (7-AAD+ and CFSE+) is shown. Data are presented as mean ± SEM for A375 compared with A375p53KO cells at 10:1 NK: tumor cell ratio (n = 3 biological replicates), *P < 0.05