Fig. 2

p53 and IFNγ signaling. A Western blots in A375 and HT144 (wt p53), and A375p53KO and RPMI7951 (p53-null) cells with or without 1 μM AMG-232 for 24 h. IRF1, p21, and β-actin (as loading control) have apparent molecular weights of 55, 21, and 42 kDa, respectively. B, C RT-qPCR of IRF 1 in A375 and A375p53KO cells (B) and in wt p53 (A375 and HT144) and p53-null (A375p53KO and RPMI7951) treated with 1 μM AMG-232 for 24 h (C). IRF1 mRNA levels were normalized to ACTB mRNA (n = 3 biological replicates, each with technical triplicates). Relative expression is shown as fold change ± SEM for treated cells compared with untreated (*P < 0.01). D Western blots in A375 and A375p53KO cells with or without 1 μM AMG-232 for 24 h and 20 μM MG-132 for the last 4 h before harvesting. IRF1, PD-L1, SOX10, MDM2, p53, p21, and β-actin (as loading control) have apparent molecular weights of 55, 55, 55, 90, 53, 21, and 42 kDa, respectively. Stripped and re-probed blots are indicated by arrows. E Western blots in A375 and A375p53KO cells treated with or without 1 μM AMG-232 for 24 h and with CHX (100 μg/ml) for the indicated times. IRF1 and β-actin (as loading control) have apparent molecular weights of 55 and 42 kDa, respectively