Fig. 3

Effect of gp90 released by T. cruzi metacyclic forms in host cell invasion. a Conditioned medium of the indicated strains, prepared in D10 medium, was analyzed by Western blot. b HeLa cells were incubated for 1 h with CL strain metacyclic forms in D10, D10 plus conditioned medium (CM) of strain 515, prepared in D10, preincubated or not with anti-gp90 mAb 1G7 or unrelated mAb 2C2. Values are the means ± SD of three independent assays performed in duplicate. Reversal of the inhibitory effect of CM by mAb 1G7 was significant (*P < 0.05). c Metacyclic forms of the indicated strains were incubated for 1 with HeLa cells and then processed for confocal immunofluorescence to visualize lysosomes (red), actin cytoskeleton (green) and nucleus (blue). The image showed a representative microscopic field of experiment under 60X objective. Scale bar = 15 μm. d HeLa cells were incubated for 30 min in D10 or in PBS⁺⁺ and processed for confocal fluorescence analysis as in (c). Note the lysosome scattering in cells incubated in PBS⁺⁺. Scale bar = 20 μm. e Metacyclic forms of the strains 515 and 1434 were incubated for 1 h with HeLa cells in D10 or in PBS⁺⁺, and then processed for Giemsa staining. Values are the means ± SD of three independent assays performed in duplicate. Cell invasion was significantly higher in PBS⁺⁺ than in D10 for strain 515 (**P < 0.005) and strain 1434 (*P < 0.01). f, g HeLa cells were incubated for 1 h with metacyclic forms of strain 515 (f) or strain 1434 (g) in PBS⁺⁺, PBS⁺⁺ plus conditioned medium (CM) of respective strain prepared in D10, preincubated or not with anti-gp90 mAb 5EG7 or unrelated mAb 2C2. Values are the means ± SD of three independent assays performed in duplicate. Significant inhibition by CM was observed in invasion by strain 515 (*P < 0.05) and by strain 1434 (**P < 0.005), which could be reverted by mAb 5E7 but not by mAb 2C2. Detection of gp90 released by each strain in D10 and in PBS⁺⁺ is shown in the upper part of the corresponding figure