Figure 1.

rEhCP112a drops TEER of Caco-2 cell monolayers without membrane disruption. (A) (-■-) rEhCP112p or (-□-) rEhCP112a or (--) BSA were incubated with Z-Phe-Arg-AMC and fluorescence was measured at OD₄₆₀ nm. Inset: WB and zymogram assays of (P) rEhCP112p and (A) rEhCP112a, the last one activated by 10 mM β-ME. Lanes 1 and 3: recognition by α-EhCP112 antibody. Lanes 2 and 4: proteolytic activity on gelatin gels. (B) Caco-2 cells were incubated with rEhCP112a, (-□-) 10, (-▴-) 20, or (-♢-) 30 μg or with (--) refolding buffer, or (-■-) rEhCP112p (10 μg) or with (-▾-) trophozoites and then, TEER was measured during 90 min. Arrow signals the time when rEhCP112a was removed (-▴-) and replaced by fresh DMEM and incubated again at 37°C, later, TEER was measured. (C) Caco-2 cells incubated for 30 min with rEhCP112a or EhCP112p (20 μg/cm²) and then stained with PI and observed by confocal microscopy. Controls included ethanol-fixed Caco-2 cells and cells incubated with the refolding buffer. ph c, phase contrast. Bar = 10 μm. (D) rEhCP112a was incubated for 5 min with (-▴-) E-64 or (-■-) α-EhCP112 antibody prior to the incubation with Caco-2 cells or with (--) refolding buffer or with (-□-) rEhCP112a, or with (-■-) rEhCP112p, or with (-▾-) EDTA, then, TEER was measured. TEER values were normalized according to the initial value given by each transwell (1000 Ω/cm²). Means and standard errors are represented for each time point of three independent assays performed by triplicate. ^*p ≤ 0.05.