Fig. 3. PD-L1 knockout confers resistance to chemotherapy-induced apoptosis and DNA double strand breaks (DSBs) that can be reversed by re-expression of PD-L1, but not its deletion mutants.

A, B. RKO cells or those with PD-L1 knockout (KO) were treated with irinotecan (CPT-11), oxaliplatin, cobimetinib, or gemcitabine (B). Protein expression of the DSB marker pH2Ax and cleaved caspase-3 were examined by immunoblotting (A, B), while apoptotic cells were quantified by annexin V labelling followed by quantification using flow cytometry (B) Statistical significance was calculated using two-way ANOVA, n=3, * p<0.05,** p<0.01. C, PD-L1 knockout MC38 cells with stable ectopic expression of human PD-L1 or its deletion mutants of the intracellular (ICD-d) or extracellular (ECD-d) domain were generated. The ability of the MC38, PD-L1 KO, or PD-L1 KO cells with re-expression of wild-type (WT) PD-L1 or empty vector (EV) to form colonies was determined using a clonogenic assay. The data is presented as mean±s.e.m. of n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA, * p<0.05,** p<0.01. D, The MC38 cell line and its derivatives were treated with CPT-11 or oxaliplatin for 24h and annexin V⁺ apoptotic cells were quantified, and expression of pH2AX and caspase-3 were determined. The data are presented as mean s.e.m. of n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA.** p<0.01.