Fig. 6. Effects of CYP5122A1 half knockout and overexpression on promastigote differentiation.

A–F CYP5122A1 overexpression alters the expression of LPG and PPG. Log phase (A, B) and stationary phase (C, D) promastigotes were subjected to Western blot analyses using the monoclonal antibody CA7AE (for LPG and PPG) or an anti-α-tubulin antibody (as loading control). A and C: whole cell lysates. B and D: culture supernatants. Relative expression levels of cellular LPG (E) and PPG (F) were determined with Ld1S WT as 1.0. G, H CYP5122A1 half knockout and overexpression reduces the expression of SHERP. Total RNA from log phase and stationary phase promastigotes were subjected to RT-qPCR analyses using primers for SHERP and 28S rDNA (as loading control). G Expression levels of SHERP in each parasite line from log phase to stationary phase using log phase levels as 1.0. H Expression levels of SHERP relative to Ld1S WT (as 1.0). I–N CYP5122A1 overexpression alters promastigote stress response. I–L Day 1 stationary phase promastigotes were incubated under various conditions and percentages of dead cells were determined by flow cytometry at the indicated times. I Complete M199 medium, 27 °C, pH7.4 (control). J Complete M199 medium, 27 °C, pH 5.0. K PBS, 27 °C, pH 7.4. L Complete M199 medium, 37 °C, pH 7.4. Day 1 stationary phase promastigotes were incubated in various concentrations of H₂O₂ (M) or SNAP (N) and percentages of dead cells were determined after 48 hours. Bars and error bars represent the means and standard deviations from three or four (E and F), three to nine (G and H), or three to six (I through N) independent repeats. Two-tailed t test without adjustment (*p < 0.05, **p < 0.01, ***p < 0.001). Source data are provided as a Source Data file.