Fig. 7

E. histolytica-induced gasdermin D cleavage regulates IL-1β secretion. a THP-1 macrophages were incubated for increasing amounts of time with 1:20 Eh to macrophage ratio. LPS (50 ng/mL) and nigericin (10 μM) stimulation for 60 min was used as a positive control (abbreviated as LN). b WT, CASP1 CRISPR/Cas9 KO, and CASP4 CRISPR/Cas9 KO macrophages were treated with Eh for 30 min. c WT and GSDMD CRISPR/Cas9 KO macrophages were incubated with Eh for 30 min. LPS (50 ng/mL) and nigericin (10 μM) stimulation for 30 min was used as the positive control. Cell supernatant from stimulated macrophages was also added to HEK-Blue™ reporter cells to detect bioactive IL-1β using the SEAP assay (d) and percentage of LDH release compared to non-stimulated cells (control) (e). Equal amounts of supernatants and lysates were loaded onto SDS-PAGE gel and immunoblot analysis was performed for GSDMD, caspase-4, caspase-1, and IL-1β in both the supernatants (SN) and lysates (LYS), and blots were reprobed for GAPDH. Data are representative of three experiments and statistical significance was calculated using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ns not significant). Bars represent ± SEM