Figure6 .

The activation of caspase-3 and ASC speck assembly are independent of but is associated with each otherThe wild-type and NLRP3−/− BMDMs planted in glass-bottom dishes were primed with 500 ng/mL LPS for 4 h, and pre-treated with or without inhibitor (2 μM MCC950, 1 μM A-804598, and 30 μM trovafloxacin for 1 h, followed by stimulation with 15 μM GOS for 3 h in the presence or absence of inhibitors. After fixation and permeabilization, the cells were stained with AlexaFluor488-conjugated anti-ASC antibody only (A) or anti-cleaved caspase-3 (C.CASP3) antibody (B,C), anti-p-MLKL antibody (E), and were stained with CF568-conjugated goat-anti-rabbit IgG, followed by AlexaFluor488-conjugated anti-ASC antibody. The nuclei were revealed by Hoechst 33342 staining. Fluorescence images were captured by fluorescence microscopy. The insets show a magnified area of one cell with an ASC speck co-localized with C.CASP3/p-MLKL punctum. White arrows indicate ASC specks co-localized with C.CASP3/p-MLKL dots which are indicated by yellow arrows. Scale bar, 10 μm. (D,F) The ratios of cells containing ASC specks were calculated by the number of cells with ASC specks relative to the total number of cells from five random fields. Data are shown as the mean±SD (n=5). ***P<0.001. ns, not significant.