Fig. 4

a c-MET amplification mediates PD-L1 moderated the activation and cytotoxic effect of T lymphocytes, and anti-PD-L1 antibodies restored the cytotoxicity of T lymphocytes. PC-9 or PC-9R cells were pretreated with anti-PD-L1 antibodies (10 μg/mL) or control IgG for an hour, then co-cultured with human PBMC for a further 72 h at an effector/target cell ratio = 6:1. The percentage of CD3⁺/CD4⁺, CD3⁺/CD8⁺ cells in the co-culture system was analyzed by flow cytometry. b IFN-γ concentration in the supernatant of co-culture systems. PC-9 or PC-9R cells were pre-treated with anti-PD-L1 antibodies (10 μg/mL) or control IgG for an hour, then co-cultured with human PBMC for further 72 h at an effector/target cell ratio = 6:1. The concentration of IFN-γ in the supernatant was detected by human IFN-γ ELISA assay (multi sciences company, EK1802). Bars indicate SD; ** P < 0.01.c-e PI3K/Akt and MAPK signaling pathways are involved in c-MET amplification mediated upregulation of PD-L1 in EGFR-TKI resistant lung cancer. c and d TAK-733 (MEK inhibitor) and PF-04691502 (dual PI3K/mTOR inhibitor) successfully inhibited the MAPK and PI3K signaling pathways. PC-9 and PC-9R cells were incubated with various concentrations of TAK-733 or PF-04691502 in concentration of 0, 0.01, 0.1, 0.3, 1 μmol/L respectively. After 12 h, the cells were harvested and analysed by western blotting and flow cytometry. e PC-9 and PC-9R cells were incubated with TAK-733 (0.3 μmol/L) or PF-04691502 (0.3 μmol/L) for 12 h, then harvested and analyzed by flow cytometry. Bars indicate SD. * P < 0.05, ** P < 0.01. All of the experiments were performed at least thrice