Fig. 4. Integrated Stress Response (ISR) pathway activation induces PD-L1 protein in NSCLC.

a, Western blot analysis of peIF2α and total eIF2α in H1944 cells expressing a control sgRNA or a sgRNA targeting UROD. b, Autoradiography analysis of newly synthesized S-35 labeled proteins in H1944 cells transfected with control or UROD siRNAs, with three biological replicates per sample. Wildtype (WT) cells treated with 20uM cycloheximide (CHX) for 1 hour prior to metabolic labeling served as positive control. c, Quantification of S-35 Met/Cys signal from (b) normalized to control siRNA cells. Error bars represent SDs from the mean, a two-tailed student t test was performed to assess statistical significance. ***p value = 0.0008. d, Western blot analysis in H1944 cells treated with Salubrinal (in μM) for 48h. e, Western blot analysis in H1944 cells treated with 100 μM Salubrinal and/or 200 nM ISRIB for 24 hours. f, Western blot analysis of peIF2α and PD-L1 in cells cultured in normoxic or hypoxic conditions for 24h and 48h. g, Quantitative real-time PCR analysis of PD-L1 mRNA in cells shown in (f). h, Western blot analysis of peIF2α, total eIF2α, and PD-L1 in cells treated with Arsenite (100uM for H1944 cells, 50uM for Calu-6 and 5uM for H358 cells) for 24h. i, Quantitative real-time PCR analysis of PD-L1 mRNA in cells shown in (h). Bar graph represents PD-L1 mRNA expression normalized to ACTIN across three technical replicates, shown as individual data points. j, Western blot analysis in eIF2α wildtype (S/S) or ser51Ala mutant (A/A) cells expressing control or Urod shRNA. Experiments in a-j were performed two independent times with similar results, and data from a representative experiment are shown.