Figure 5. Caspase-11 autoprocessing mediates noncanonical inflammasome assembly.

(A) Schematic diagram indicating co-transfection combinations used in (B–D). Unlabeled wild-type (WT), catalytically inactive (C254A), or non-cleavable (D285A) caspase-11 constructs were transfected into HEK293T cells at increasing doses, together with a fixed dose of catalytically inactive (C254A) mCherry-tagged caspase-11. (B) 12 hr post-transfection, whole-cell lysates were harvested and immunoblotted for mCherry or β-actin as loading control. (C) 18 hr following transfection, cells were imaged by fluorescence microscopy. Nuclei (blue) are stained with Hoechst, white arrows denote caspase-11 specks, scale bar = 10 μm. (D) Speck formation was quantified as percentage of mCherry-expressing cells containing at least one speck. (E) HEK293T cells stably expressing human gasdermin D (hGSDMD) were transiently transfected with a fixed dose of empty plasmid (Veh) OR mCherry-tagged C254A caspase-11, plus increasing doses of unlabeled WT, C254A, or D285A caspase-11 constructs. Cytotoxicity was measured as percent lactate dehydrogenase (LDH) release 18 hr post-plasmid transfection. All error bars = mean ± SEM of triplicate wells (800–900 cells per well); representative of three independent experiments. Dose–response curves were plotted by least-squares nonlinear regression ([Log₂(agonist) vs. response (three parameters)]; Y = Bottom + (Top-Bottom)/(1 + 10^(LogEC50-X)); R² indicated). Two-way ANOVA (highest doses) with Sidak’s multiple-comparison test, ns, not significant, ***p<0.001, ****p<0.0001.