Fig. 3

Regulation of PD-L1 expression by PKM2 in a lung adenocarcinoma cell line. PKM2 expression in A549 lung adenocarcinoma cells transfected with PKM2-shRNA and scramble-shRNA lentiviruses. Seventy-two hours later, the cells were subjected to puromycin selection (1 μg/mL) for 2 weeks. Subsequently, the cells were collected for qRT-PCR and protein analysis. Quantification of both qRT-PCR and Western blotting data showing that the knockdown of PKM2 with shRNA-1 and shRNA-2 markedly suppressed PKM2 mRNA levels (a) and total protein expression (b) compared with those with scrambled control. (c) Western blotting of total PD-L1 protein levels in PKM2-shRNA-1-, PKM2-shRNA-2- and scramble-shRNA-transfected lung adenocarcinoma cell lines. (d) Flow cytometric analysis of cell surface PD-L1 levels in PKM2-shRNA-1-, PKM2-shRNA-2- and scramble-shRNA-transfected lung adenocarcinoma cell lines. PKM2-shRNA1, blue zone; PKM2-shRNA2, green zone; scramble-shRNA, red zone; isotype control, grey zone. Unpaired t tests were used to calculate the two-sided P-values. ^*P < 0.05, ^**P < 0.01. Error bars in the bar charts represent standard deviation