Cat S inhibition causes defective autophagy flux in macrophage in the tumor microenvironment. (A) WT or Cat S-/- macrophages were transfected with Cherry-GFP-LC3, then cocultured with SL4 cells for 48 hrs and serum-starved for at least 12 hrs before co-culture. Confocal fluorescence was used to image autophagosomes (red and green foci) and autolysosomes (red-only foci). Scale bars = 10 μm. Three independent experiments were performed. (B) WT or Cat S-/- macrophages expressing the GFP-LC3 reporter construct were seeded in plastic wells, then recognition and counting by fluorescence image analysis using the KineticScan HCS Reader. Data are mean ± SEM of 3 independent experiments. **, P<0.01. NS indicates not significant. (C) WT or Cat S-/- macrophages were cocultured with SL4 cells and serum-starved for at least 12 hrs before co-culture, and WT macrophages in the presence or absence of Cat S inhibitor Z-FL-COCHO (10 μmol/L). WT or Cat S-/- macrophages were loaded with 10 μg/mL DQ-Green BSA for 15 min, washed twice and incubated in media for the indicated times, then subjected to flow cytometry analysis. Background (gray peak) represents samples without the DQ-Green BSA loading. Three independent experiments were performed. (D) WT or Cat S-/- macrophages were placed on coverslips as above in C, and incubated in media containing DQ-Green BSA (10 μg/mL) for 15 min. Cells were washed twice with PBS and incubated in media for the indicated times. Cells were fixed and the fluorescent degradation products of the DQ-Green BSA in lysosome were imaged using confocol images analysis. Scale bars = 5 μm. Dotted lines indicate the cell margin. Three independent experiments were performed.