CYP5122A1, a Novel Cytochrome P450 of Leishmania Figure 3. Subcellular localization of CYP5122A1. A: Western blot of aqueous (Aq) and detergent (Det) phases of Triton X-114 extracts of Leishmania promastigotes probed with anti-CYP5122A1 antibody. B: Photomicrographs of Leishmania donovani promastigotes stained with anti- CYP5122A1 antibody and organelle specific markers. BIP & ER Tracker Blue-White DPX, ER marker; MitoTracker Red, mitochondrial marker; GAPDH, glycosome marker. a: Nomarski; b: cells stained with anti-CYP5122A1 antibody; c: cells stained for organelle specific marker; d: merge of b and c; e: mask of colocalization for d. Scale represents 10 mm. doi:10.1371/journal.pone.0025273.g003 regulation of CYP5122A1 at doses of 10–40 mM (Fig. 8D, i and ii) with maximal induction at 24 h with 20 mM of miltefosine (Fig. 8D, iii). Multiple drugs act via generation of ROS [24–25]. ROS levels measured after miltefosine exposure showed increased ROS in both the WT and the HKOs, generation in the HKOs being higher (Fig. 8E). Scavenging this ROS with the antioxidant N-acetyl cysteine (NAC, 20 mM), resulted in a substantial reduction of cell death in the HKOs (cell death %, control, 260.3; HKO, 3564.0; HKO+NAC, 1561.0, n = 3) suggesting that ROS generation was linked to drug efficacy. Therefore, the above observations strongly imply that a normal complement of CYP5122A1 is required by the Leishmania parasite for defense against drugs. PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25273