FIGURE 2.
Attenuated enzymatic activity of caspase-1 increases fluorescence intensity and size of the pyroptosome. iBMDMcasp1/11-ko cells expressed GFP-ASC in combination with either Ch-WT or Ch-C284A. Caspase-1 activation was induced by 3 h of priming with 5 μg/ml LPS, followed by 10 μm nigericin. Fluorescence life cell imaging was initiated subsequently. A, bright-field (gray) and Hoechst (blue nuclei) channels were merged with either the GFP channel (green, first and third rows), the cherry channel (red, second and fourth rows), or both (Merge). Representative images were chosen between 0–16 min (Ch-WT) or 0–11 min (Ch-C284A), respectively, after speck formation (arrowheads). B and C, cells expressing both fluorescent proteins were analyzed for speck formation. Graphs indicate mean values of maximum fluorescence intensities of Ch-WT or Ch-C284A specks (red) or GFP-ASC specks (green). First speck formation was set as the starting point (0 min) and assessed for each pyroptosome individually. Intensity levels 10.5 min before speck formation (−10.5 min) represent the background signal from the cytosol. DMSO as loading control was excluded from E for matters of clarity but included in F. Speck intensities after 158.5 min were chosen for statistical analysis in F (n ≥ 64 of three independent experiments). D–F, cell lines (as labeled in B and C) were treated as indicated. Ethanol served as a loading control for nigericin and DMSO as a loading control for Ac-YVAD-CMK (YVAD). D, in Western blots from supernatants (SN), we detected the pro-form and active form of IL-1β (33 and 17.5 kDa, respectively) as well as the active caspase-1 p10 subunit (Casp1, 10 kDa). E, IL-1β release into supernatants was measured with cytometric bead arrays (n = 2). F, lactate dehydrogenase (LDH) release into supernatants represents cell death (n = 6). G, speck sizes were measured in cells expressing both mCherry-caspase-1 (Ch-Casp-1) and GFP-ASC (n ≥ 31 of three independent experiments). Statistical significance was determined by Mann-Whitney test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant. Standard errors of the mean are indicated.