Clathrin inhibition blocks phagosome resolution. (A) Single-cell live-cell imaging time series of RAW cell expressing CLC-GFP showing internalized IgG-opsonized beads at 3 h after phagocytosis. Color scale indicates the fluorescence intensity of clathrin GFP. Time interval between frames is 10 s. Asterisk indicates internal beads, and green arrows indicate accumulation of clathrin around phagosomes. Scale bars: 5 µm. (B) RAW cells internalized mRFP1–E. coli for 1 h before treatment with ikarugamycin. Live-cell imaging commenced after uptake (0 h) or >6 h after phagocytosis. (C) Intact phagosomes and PDVs per cell were quantified as puncta (particle number) for experiments displayed in B and normalized to 0 h. Data are shown as mean ± SEM of 15 images per control/treatment across 3 independent experiments, where each image display 8–29 cells. Control and inhibitor-treated cells at each time point were compared statistically using two-way ANOVA with Sidak’s post hoc test (*, P < 0.05). (D) RAW macrophages internalized mRFP1–E. coli, were chased for 1 h, and then were treated with either vehicle or 10 µM Pitstop 4 or 6 h after phagocytosis. (F) Primary macrophages were presented with mRFP1–E. coli and were treated with vehicle, Pitstop, or ikarugamycin after maturation. Cells were fixed and imaged 3 h after phagocytosis. Dashed lines show cell contours in B, D, and F. Scale bars: 10 µm (B, D, and F). (E and G) PDVs/cell were quantified in 60 cells per condition across 3 independent experiments and compared statistically using one-way ANOVA with Dunnett’s multiple comparison test. (*, P < 0.001; **, P < 0.0001).