Figure 5

Lysosomal leakage of cathepsin B mediates CATH-2-induced NLRP3 activation in LPS-primed macrophages. Cells were primed with LPS for 3 h and then Baf.A1 (100 nM) and Cyto.B (10 μM) were added for 1 h incubation prior to CATH-2 treatment for a further 21 h. ELISA was used to determine cytokine secretion of IL-1β (A) and IL-6 (B). Confocal microscopy images of cells loaded with Alexa 488 Dextran (green) treatment with LPS and CATH-2, with dashed white arrows indicating fluorescent signal in lysosomes and solid white arrows indicating lysosomal leakage (C). Furthermore, cells were pretreated with cathepsin B inhibitor (CA-074-Me, 20 μM) and cathepsin D inhibitor (pepstatin A, 20 μM) for 1 h. Subsequently, cells were primed with LPS for 3 h and then CATH-2 (5 μM) was added for an additional 21 h. ELISA was used to determine cytokine secretion of IL-1β (D). A representative image of the Western blot assay is present to show protein expression including IL-1β, caspase-1, pro-IL-1β, pro-caspase-1 and β-action (E); Additionally, after 6 h incubation, immunofluorescence staining was performed using primary antibodies anti-NLRP3 and anti-ASC as well as secondary antibodies Goat anti-mouse IgG (H&L) Alexa fluor 488 and Goat anti-rabbit IgG (H&L) Alexa fluor 594. Representative images of ASC, ASC specks (white arrows), NLRP3 and cell nucleus (F). Data from figure A are represented as mean ± SEM of three independent experiments of triplicate samples per experiment. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005.