Fig 5. Cruzipain increases T. cruzi invasion through TGF-β activation.
(A) Vero cells (1 × 105 cells per well) were infected with trypomastigote forms of T. cruzi (Y strain) that were added to cultures in a mixture containing purified cruzipain (CZP, 10 μg ml-1) or anti-TGF-βantibodies (10 μg ml-1) or FMK (50 μM) or CZP (10 μg ml-1) in a combination with anti-TGF-β antibody (10 μg ml-1) or FMK (50 μM). (B) Parasites were pre-treated with different combinations of compounds before addition to Vero cell culture: trypomastigotes were treated or not with Z-Phe-Ala-FMK (FMK, 50 μM) for 30 min at 37°C and then incubated or not with recombinant latent TGF-β (LTGFβ 100 ng ml-1) or with LTGFβ (100 ng ml-1) plus CZP (10 μg ml-1) for 1 h at 37°C. Vero cells were then infected with pre-treated parasites. Cells were fixed 48 h post infection and stained with Giemsa. Representative images are shown (C to H). Arrows indicate infected cells. The percentage of infected cells was determined by counting 400 cells per slide in two distinct slides. Data are the mean ± SD (*P < 0.05, ** P < 0.01, *** P < 0.001 when compared with controls and # P < 0.05, ## P < 0.01, ### P < 0.001 when compared with CZP (A) and LTGFβ (B). n = 3.