Fig 6. Cathepsin B and K⁺ efflux both function as signals 2 in H. capsulatum-stimulated inflammasome activation.

Wild type BMDCs were pretreated with (A) cathepsin B activity inhibitor (Ca-074 ME, 25 μM), phagosome acidification inhibitor (bafilomycin A1 (BA1), 250 nM), (B) extracellular KCl (50 mM) and NaCl (50 mM), K⁺ channel inhibitor (glibenclamide (Gliben), 50 μM), (C) ROS inhibitor (apocynin (Apo), 10 μM) and ROS scavenger (N-acetyl cysteine (Nac), 20 mM) for 1 h before stimulation with H. capsulatum. Cell-free supernatants were harvested at 18 h after stimulation. Secreted IL-1β was quantified by ELISA (n = 3). (D) BMDCs were treated with DQ ovalbumin for 1 h before stimulation with (Hc) or without (ctrl) H. capsulatum at MOI of 1. Cells were collected at 1 h after stimulation before cytospun on microscope slides. Cells were fixed and stained for CD11c (red). The nuclei were stained by Hoechst reagent (blue). Cells were viewed under confocal microscope. White arrows point to ingested-H. capsulatum yeasts in DIC/Hoechst field. (E) BMDCs (2 × 10⁶) were treated with or without Ca-074 ME (25 μM) and bafilomycin A1 (250 nM) before stimulation by H. capsulatum at MOI of 20 for 18 h. Cell lysates and supernatants were analyzed for cathepsin B activity (n = 3). (F) Wild type BMDCs were treated with Ca-074 ME (10 and 25 μM) before stimulation with H. capsulatum. Cell lysates and supernatants were analyzed for inflammasome components by Western blotting. Error bars indicate standard deviation of the mean. One representative of three (A, B and C) and two (D, E and F) independent experiments is presented. * p < 0.05, ** p < 0.01, *** p < 0.001 [2-tailed t-test (A, B and C); one-way ANOVA with Tuckey post-hoc analysis (E)].