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. 2020 Sep 30;21(19):7229. doi: 10.3390/ijms21197229

Figure 5.

Figure 5

pH sensitivity was conferred upstream of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) oligomerization. (a) Detection of caspase-1 in supernatants and pro-caspase-1, NLRP3, and ASC in lysates with Western blot after iBMDMs were treated overnight with 200 μg/mL Pam3CSK4 and 1 h with 10 μM nigericin or buffers alone. (b) Detection of ASC specks (red aggregates) in immunofluorescently labelled WT iBMDM with confocal microscopy after cells were primed with 100 ng/mL LPS, followed by caspase-1 inhibition by 30 min treatment with 100 μM acYVAD and 1 h incubation in buffers with various pH values alone or containing 10 μM nigericin. Nuclei were stained with DAPI (blue). Scale = 40 μm. Images were quantified by determining the number of specks/number of nuclei ratio for 6 z-stack image frames for each experimental condition. (c) IL-1β levels by ELISA in supernatants of ASC−/− iBMDMs after overnight priming with 100 ng/mL LPS and subsequent incubation in buffers with various pH values alone or containing Saty flagellin in DOTAP transfection reagent.