Fig. 4. : correlation between the expression of surface gp63-like and cruzipain-like molecules and the interaction of Phytomonas serpens with salivary glands of Oncopeltus fasciatus. (A) Promastigotes were pre-treated or not (control) with the IC50 value of the calpain inhibitor MDL28170 for 24 h; then, paraformaldehyde-fixed cells were incubated in the presence of anti-gp63 and anti-cruzipain antibodies and analysed by flow cytometry. (B) Parasites were pre-treated or not (control) with the IC50 value of MDL28170 for 24 h. Following interaction for 1 h, the salivary glands were washed and homogenised, and the released trypanosomatids were counted in a Neubauer chamber. The results are shown as the mean ± standard deviation of three independent experiments. (C) Flow cytometry analysis showing calpain-like proteins (CALPs) cross-reactive to anti-Dm-calpain antibody, the gp63-like and cruzipain-like proteins expressed in P. serpens cultured in Warren medium and in parasites recovered from the interaction process with the salivary glands. In (A) and (C), data are expressed as the percentage of fluorescent cells (% FC) and also the mean of fluorescence intensity (MFI) levels. Representative data of the analysis of 10,000 cells from three experiments are shown. The stars and the asterisks highlight significant different values for % FC and MFI levels, respectively, compared to control in each system (p < 0.05). In (B), the results are shown as the mean ± standard deviation of three independent experiments and the star denotes significant difference compared to control (p < 0.05).