Fig. 5: Depletion of PD-L1 by RNAi reduces NHEJ activity and enhances HR activity.
a H460 cells were transfected with Ctrl siRNA, PD-L1 siRNA, 53BP1 siRNA, RIF1 siRNA or BRCA1 siRNA, followed by western blot. b, c NHEJ activity was analyzed by FACS using BDTM FACSCanto II in H460 cells expressing Ctrl siRNA, PD-L1 siRNA, 53BP1 siRNA, or RIF1 siRNA. Y axis represents the recovery rate of GFP from I-SceI-induced DSBs in EJ5-GFP reporter. X axis represents 7-AAD (a nucleic acid dye) staining, which was used for the exclusion of nonviable cells for quality control in flow cytometric analysis. Error bars represent ± s.d., n = 3 per group. ***P < 0.001, by two-tailed t test. d H460 cells or H460 PD-L-1 knockout (KO) cells were treated with IR (4 Gy), followed by immunofluorescence co-staining of γH2AX with anti-53BP1, RIF1 or FAM35A, respectively. e, f HR activity was analyzed by FACS using BDTM FACSCanto II in H460 cells expressing Ctrl siRNA, PD-L1 siRNA, 53BP1 siRNA, BRCA1 siRNA+ PD-L1 siRNA or BRCA1 + 53BP1 siRNA. Y axis represents the recovery rate of GFP from I-SceI-induced DSBs in DR-GFP reporter. X axis represents 7-AAD (a nucleic acid dye) staining, which was used for the exclusion of nonviable cells for quality control in flow cytometric analysis. Error bars represent ± s.d., n = 3 per group. *P < 0.05, **P < 0.01, by two-tailed t test. g H460 cells or H460 PD-L-1 knockout (KO) cells were treated with 4 Gy of IR at 2 Gy/min dose rate, followed by immunofluorescence co-staining of γH2AX with pRPA2 or RPA70, respectively. Source data are provided as a Source Data file.
