Fig 2. Induced ectopic expression of TcUBP1-GFP in early-log phase epimastigotes promotes the development of metacyclic trypomastigotes features.

(A) Immunofluorescence of infective metacyclic trypomastigotes showing staining with anti-TS-SAPA antibody, and non-infective epimastigotes showing no staining. (B) Western blot of epimastigotes (E) and metacyclic trypomastigotes (MT) protein extracts with the anti-TS-SAPA antibody. An extract from cell-derived trypomastigotes (T) served as a positive control. Tubulin immunoblot was used as loading control. (C) Staining of TS-SAPA by immunofluorescence in induced (10 days) or uninduced early-log epimastigotes expressing TcUBP1-GFP or GFP. DNA was stained with DAPI. Images are representative of 3 independent experiments. (D) Western blot of uninduced or induced epimastigote protein extracts ectopically expressing TcUBP1-GFP. As a positive control for the anti-TS-SAPA antibody we used a protein extract from cell-derived trypomastigotes (T). Tubulin blot served as a loading control. Image is representative of 4 independent experiments. Scale bars, 5 μm. (E) Survival of epimastigotes to lysis by human complement. TcUBP1-GFP uninduced or induced parasites were incubated in PBS supplemented with 10% fresh human serum for the defined time course. Microscopic analysis was performed at each time point without fixation, only motile parasites were considered alive. **P< 0.01 by Two-way Anova- test.