Fig. 1

PD-L1 is dynamically maintained on the plasma membrane of cells. A Representative confocal images of primary human tumor cells immunostained for plasma membrane (PM) and total PD-L1. Red: PD-L1. Blue: Hoechst. B Quantification of percentage of PM PD-L1 in primary human tumors. Each data point is a patient sample color coded for known oncogenes. C, D Quantification of C percentage of PM PD-L1 and D total PD-L1 protein expression in human lung cell lines via immunofluorescence imaging. Cell lines in blue and red harbor mutant KRAS and mutant EGFR respectively. E Representative confocal images of BEAS-2B with or without IFNγ treatment and immunostained for PM and total PD-L1. Red: PD-L1. Blue: Hoechst. Sum intensity of z-stacks is projected. Fluorescence intensity is equally scaled across panels. F Quantification of PM localization of PD-L1 in BEAS-2B cells treated with IFNγ. Unpaired student’s t-test. G Schematic of PD-L1 exocytosis assay. Live cells are incubated with unlabeled PD-L1 antibody. Cells are fixed, permeabilized and stained with fluorescently labeled secondary antibody depicted as a star. H PD-L1 steady state exocytosis in BEAS-2B cells. The line is a fit of the data to one-phase association exponential equation, N = 3. I PD-L1 PM to total ratio with growth factors treatment or Akt inhibition in BEAS-2B cells. One-way ANOVA and post-hoc Tukey test comparing each condition to -serum. J PD-L1 PM to total ratio in cell lines treated with vehicle (DMSO) or Akt inhibitor for 4 h. One sample t-test. K PD-L1 PM to total ratio in IFNγ treated BEAS-2B cells incubated with vehicle (DMSO) or Akt inhibitor. One sample t-test. L PD-L1 PM to total ratio in CMTM6 knocked-down (KD) IFNγ-treated BEAS-2B cells with vehicle (DMSO) or Akt inhibitor. Except panel B and H, each data point is an experiment. All error bars = SEM. * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, # = 0.058