Fig. 5

Cell surface PD-L1 as a function of ATG5 status. a A375 p53-wt and A375 p53-null cells were treated with 10 μM MG-132 for the indicated times. PD-L1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control antibody have apparent molecular weights on Western blots of 50 and 37 kDa, respectively. b A549-Atg5-wt cells (wt) were processed using gene editing (CRISPR-Cas9) [32] to create mutant A549-Atg5-null cells (null). Atg5-GFP was introduced into A549-Atg5-null cells to create a partial Atg5 rescue phenotype (resc). Both PD-L1 and GAPDH were detected in these cells. Representative flow cytometric graphs of c A549-Atg5-wt, d A549-Atg5-null and e A549-Atg5-resc. Cell surface PD-L1 was measured using APC-conjugated antibody. Results represent the mean ± SD of technical triplicates, each with 30,000 counted cells. f Normalized mean fluorescent intensity (MFI) of PD-L1 derived from FACS on c - e. Statistical analysis was performed by one-way ANOVA with Bonferroni post hoc test, * p < 0.05, *** p < 0.001. g PD-L1 (upper row) and CD276 (lower row) were measured after treatment with 10 μM chloroquine for the indicated times by FACS using APC- and FITC-conjugated antibodies, respectively