(A) Representative dot plot of PD-L1 (x-axis) and prostasin expression (y-axis) in Calu-3 cells treated with IFNγ (100 ng/ml for 24 h). Red: unstained cells. Green: secondary antibody (anti-rabbit-Cy2) and isotype IgG-APC. Magenta: prostasin antibody-labeled cells. Orange: PD-L1-APC antibody-labeled cells. Sky blue: prostasin and PD-L1 antibodies double-labeled cells. (B) Representative dot plot of prostasin expression in Calu-3 sublines. CC (red): Calu-3 control subline with scrambled gRNA, KO (green): Calu-3 knockout subline with prostasin-specific gRNA, V (magenta): Calu-3 subline with vector alone, P (orange): Calu-3 subline overexpressing the wildtype prostasin, M (sky blue): Calu-3 subline overexpressing an active-site mutant prostasin, G (blue): Calu-3 overexpressing an active prostasin without the GPI anchor. (C,D) Histogram of PD-L1 expression in Calu-3 sublines without IFNγ treatment (C) or with IFNγ treatment (D). (E) MFI of PD-L1 (filled boxes) and isotype antibody (open boxes) from duplicate settings were quantified and presented in bar graphs. Data are presented as mean ± SD. ANOVA P<0.05, * denotes P<0.05 as compared with the vector control (V).