Fig. 2

E. histolytica-induced caspase-4 activation parallels caspase-1 requiring live parasite and contact via Gal-lectin and EhCP-A5 and involves cellular perturbations. a THP-1 macrophages were incubated with different preparations of Eh including live Eh, whole lysates, or with membrane (mem. comp.) or cytoplasmic fractions (cyto. comp.) of equal amounts of Eh for 60 min. b THP-1 macrophages were pretreated for 5 min with 55 mM D-galactose (Gal), or glucose (Glu) as an osmotic control and then incubated with Eh for 60 min at a 1:20 ratio (optimal dosage from Fig. 1a). c THP-1 macrophages were incubated with Eh, E-64 treated Eh, and different trophozoites, Eh deficient in CP5 (EhCP-A5⁻) and Eh deficient in amoebapore as the vector control (EhAP-A⁻) at a 1:20 for 60 min. THP-1 macrophages were pretreated with exogenous d potassium chloride (KCl) or e diphenyleneiodonium chloride (DPI) for 1 h and then incubated with Eh for 60 min. f THP-1 macrophages were pretreated with 300 μM oxidized ATP (oATP) for 2 h and then incubated with Eh for 30 min. 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, caspase-1, IL-1β in both the supernatants (SN) and lysates (LYS). Blots were reprobed for GAPDH. Western blots are representative of three independent experiments