CYP5122A1, a Novel Cytochrome P450 of Leishmania Figure 5. Morphology and biochemistry of HKO parasites. A: Photomicrographs of L. donovani WT and CYP5122A1 HKO cells during in vitro growth cycle. Scale 5 mm. B: Growth pattern of WT and HKO parasites in vitro over a period of 6 days in culture. Mean 6 SE, n = 3. C: Comparison of average constitutive levels of intracellular ROS in WT and HKO parasites. Mean 6 SE, n = 3, * P#0.05. D: DYm measured by the potentiometric probe JC-1. Mean 6 SE, n = 3; * P#0.05. E: Bar graph showing the basal level of ATP produced by the WT and the HKO cells. Mean 6 SE, n = 3; *P#0.05. F: Bar graph shows mitochondrial Ca2+ levels. Mean 6 SE, n = 3; * P#0.05. doi:10.1371/journal.pone.0025273.g005 Complementation of CYP5122A1 rescues phenotype HKO parasites were complemented with WT copy of CYP5122A1 through episomal expression with the pXG-GFP+2 vector (expressing the CYP5122A1 gene). Fig S6A shows the presence of mRNA for CYP5122A1 in the transfected cells and the presence of the fusion protein was demonstrated by western blot and flow cytometry (Fig. S6B, C). The complementation resulted in rescue of morphology from shorter HKO cells to elongated forms resembling WTs (Fig. S6D). When drug susceptibility of the HKO parasites was compared to the parasites PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25273