NADPH oxidase and V-ATPase are both required for LAP. (a) Confocal DIC and GFP-hLC3A images of OPZ phagocytosis in RAW264.7 cells treated with DPI (5 μM), GSK (10 μM), BafA1 (100 nM), or ConA (100 nM). Cropped insert showing pseudo-colored GFP fluorescence intensity. Asterisks denote phagosomes. Scale bar, 5 μm, cropped boxes, 7 μm. (b) Quantification of GFP-hLC3A intensity at phagosomes in cells treated as above. Data represent the mean of individual phagosome measurements from three independent experiments (red triangle, green square, blue circle); ****, P < 0.0001; ***, P < 0.0007, one-way ANOVA followed by Dunnett’s T3 multiple comparison test. (c) Representative confocal DIC images of NBT/formazan deposits in phagosomes from control, DPI, GSK, BafA1, and ConA treated RAW264.7 cells. Inserts are zoomed in phagosomes. Scale bar, 5 μm, cropped boxes 7 μm. (d) Luminol measurements of ROS during OPZ phagocytosis in RAW264.7 cells treated with DPI or BafA1. Data represent the mean ± SEM from three independent experiments. AU, arbitrary unit.