Fig. 8

E. histolytica-induced IL-1β secretion is dependent on caspase-4 interaction with caspase-1. a COS-7 cells were transfected with pro-caspase-1 (pC-1, 100 ng), pro-caspase-4 (pC-4, 10 ng), pro-IL-1β (pIL-1β, 1 μg) plasmids, or GFP (pGFP, 1 μg) as a control and stimulated with Eh (1:2) for 30 min or b LPS (50 ng/mL for 2 h to prime) and ATP (3 mM for 60 min) abbreviated LA, served as the control for IL-1β release. c Cell supernatant from stimulated macrophages was added to HEK-Blue™ reporter cells to detect bioactive IL-1β using the SEAP assay. d Protein expression of caspase-4, caspase-1, IL-1β was compared in COS-1 and COS-7 lysates to indicate basal expression and rationale for usage of COS-7 cells. Cell supernatant was TCA precipitated and cells were washed and lysed. Equal amounts of lysates were loaded onto SDS-PAGE gel and immunoblot analysis was performed for caspase-4 and -1 in the supernatants (SN) and along with IL-1β in the lysates (LYS). Blots were reprobed for GAPDH. Data are representative of three experiments and statistical significance was calculated with ANOVA and Bonferroni’s post-hoc test (**p < 0.01, ***p < 0.001, ns not significant). Bars represent ± SEM