Fig 3.

Pfmspdbl2 mutations in the absence of CNV affect parasite responses to halofantrine and structurally related antimalarials. (A) Southern blot assays of Dd2 wild-type (WT) and Pfmspdbl2 allelic replacement parasites where the endogenous genome locus has been replaced with HA-tagged versions of the DR allele (S591 cloned from Dd2) or the AC allele (C591 cloned from Senegal P26.04). Digestion of the wild-type, untagged Pfmspdbl2 locus with ClaI and NotI generates a single band of 5.0 kb, while digestion of the HA-tagged integrated locus generates two bands of 4.1 and 8.0 kb. (B) Western blotting of ring, trophozoite (Troph), and schizont (Schiz) stage cultures of the DR line with anti-HA and anti-LDH antibodies shows expression of HA-tagged MSPDBL2 in schizonts. (C) Immunofluorescence of a representative schizont and representative merozoites of the DR line with anti-HA antibodies (green) and DAPI staining (blue). (D) Drug responses of DR and AC lines to halofantrine (HFN), mefloquine (MFQ), and lumefantrine (LUM) measured by tritium-labeled hypoxanthine incorporation. (E) Drug responses of DR and AC lines to chloroquine (CQ), artemisinin (ART), and atovaquone (ATV). The values shown are means ± standard deviations of at least three separate assays run on different days. *, P < 0.05 (two-tailed unpaired t test).