in vitro

Raw data from T. cruzi infection assay

Western Blot CZP

Western blot assay for cruzipain observatio

Inhibition of cysteine proteases and TGF-β activities impairs <i>T</i>. <i>cruzi</i> infection and replication.

<p>(A) Different concentrations of Z-Phe-Ala-FMK (1 to 100 μM) or (C) neutralizing anti-TGF-β antibody (1 to 10 μg ml^-1) were added to Vero cell cultures and their effects over the processes of host cell invasion by trypomastigotes of <i>T</i>. <i>cruzi</i> (Y strain) and (B and D) intracellular parasite growth were evaluated. Results are expressed as the percentage of infected cells or as the ratio of parasite number per infected cell, both determined by counting 400 cells per slide in two distinct slides from three independent experiments. Data are the mean ± SD. (*<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001). n = 3.</p

Effect of Z-Phe-Ala-FMK, a cysteine protease inhibitor, on TGF-β activation.

<p>Active TGF-β was measured by ELISA after incubation of (A) parasite lysate equivalent to 2.5 × 10⁶ epimastigotes or (B) 100 μg ml^-1 of purified cruzipain for 1 h at 28°C with or without the cysteine peptidase inhibitor, Z-Phe-Ala-FMK, at different concentrations (0.1 to 10 μM). Results are expressed as fold induction, recombinant latent TGF-β (LTGFβ) incubated alone for 1 h at 28°C has been taken as 1 (white bars). Data are the mean ± SD. (*<i>P</i> < 0.05, ** <i>P</i> < 0.01). n = 3.</p

Transfected parasites overexpressing chagasin prevent TGF-β activation.

<p>(A) Live transfected Dm28c epimastigotes overexpressing chagasin (pCHAG), empty vector (pTEX) or wild type Dm28c (WT) were incubated with latent TGF-β (100 ng ml^-1) (LTGF-β) for 1 h and the activated TGF-β was measured by ELISA. Insert: Dm28c epimastigotes that were not incubated with LTGF-β do not present significant levels of active TGF-β (pg/ml) as compared to the levels of activated LTGF-β incubated for 1h at 37°C—represented by a white bar in Fig 3A). (B) Expression pattern of cruzipain in lysates from <i>T</i>. <i>cruzi</i> Dm28c epimastigotes wild type (WT), transfected with empty vector (pTEX) or overexpressing chagasin (pCHAG) by Western blot. PageRuler Plus Prestained Protein Ladder (Thermo Scientific) was used as the molecular mass marker (MW). Results are expressed as fold induction, recombinant latent TGF-β incubated alone for 1 h at 28°C has been taken as 1. Data are the mean ± SD (*<i>P</i> < 0.05, ** <i>P</i> < 0.01 when compared with WT and ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01 when compared with pTEX). n = 3.</p

Cruzipain increases <i>T</i>. <i>cruzi</i> invasion through TGF-β activation.

<p>(A) Vero cells (1 × 10⁵ cells per well) were infected with trypomastigote forms of <i>T</i>. <i>cruzi</i> (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 (*<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 when compared with controls and ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01, ^###<i>P</i> < 0.001 when compared with CZP (A) and LTGFβ (B). n = 3.</p