FIGURE 5.

The lysosome is required for lipotoxic inflammasome activation. A, pMACs were loaded with tetramethylrhodamine (TMR)-dextran to label lysosomes followed by stimulation with BSA-PBS or palmitate (palm)-LPS for 16 h. The cells were stained with caspase-1 FLICA and analyzed by flow cytometry. The percentage of cells in each quadrant is indicated. B, the cathepsin B inhibitor CAO74 was added at the indicated doses, and IL-1β release following palmitate-LPS challenge was assessed at 20 h. C and D, macrophages were incubated with CAO74 (black bars) or another cathepsin B inhibitor, Z-FA (50 μm, hatched bars), during stimulation with BSA-PBS or palmitate-LPS for 20 h. IL-1β (C) and TNFα (D) concentrations in the supernatant were determined by ELISA. E, pro-IL-1β levels in palmitate-LPS-treated pMACs ± CAO74 (10 μm) were determined at 16 h by Western blotting. Bar graphs indicate the mean ± S.E. for a minimum of three experiments, each performed in triplicate. *, p < 0.05 for vehicle (veh) versus inhibitor. ns, not significant.