FIGURE 2.
Cathepsin B and NLRP3 interact in monocytes/macrophages under inflammasome activation. (A,B) BMDMs were primed with 300 ng/mL of LPS for 3 h. Cells were then treated with nigericin (30 min – 40 μM), ATP (30 min – 5 mM), SiO2, CPPD or MSU (6 h – 100 μg/mL) and stained with anti-NLRP3 and anti-Cathepsin B for PLA experiments. Representative images (A) and percentages of cells with fluorescent dots were evaluated (B). (C) Primed BMDMs were treated with nigericin (30 min – 40 μM), stained with indicated antibodies and assayed for PLA. (D) LPS primed THP-1 cells treated or not with ATP (30 min – 5 mM) were lysed, immunoprecipitated with anti-NLRP3 or anti-Cathepsin B antibodies and analyzed by western blot. (E,F) BMDMs treated as indicated were stained either with anti-calreticulin, anti-Tom20 or anti-Lamp-1 and specific secondary antibodies (green), with DAPI (blue) and with anti-NLRP3 and anti-Cathepsin B antibodies and assayed for PLA (red). Representative images are shown. Data represent either one representative experiment or the mean of at least three independent experiments ± SD. Statistics compared untreated cells with treated cells. *p < 0.05, **p < 0.01, no symbol, not significant.