Fig. 3

Iron could completely rescue the growth defects of PyDMT1 hypomorph in vitro. a Schizont maturation of Pydmt1-C2 rescued with 50 μM FeSO4. One hundred micromolars ascorbic acid was added to prevent oxidation/maintain Fe²⁺ status. After 18 h incubation (2 p.m. to 8 a.m., at the atmosphere of 90% N₂, 5% O₂, and 5% CO₂), most parasites in early ring stage developed to mature schizont (one hemozoin is surrounded with merozoites before release). Merozoites were counted under a microscope with blood smear. Shown are values from individual parasites as well as the mean values. One-way ANOVA and Tukey’s multiple comparison test. ****P < 0.0001. N = 80 parasites from three independent experiments. b Merozoite numbers of Pydmt1-A1 rescued with 50 μM FeSO4 in the presence of 100 μM ascorbic acid (N = 80 parasites from three independent experiments, one-way ANOVA and Tukey’s multiple comparison test. ****P < 0.0001). c A representative histogram showing the Calcein-AM fluorescence intensities of the control PyDMT1-HA(N1) and the mutant Pydmt1-A1(A1) parasites. FITC-A in x-axis represents the Calcein-AM fluorescence intensity. DIP (2,2-dipyridyl) is an iron chelator and the differential ΔMFI after iron chelation is commonly used to measure the labile iron pool. d The LIPs of PyDMT1-HA and Pydmt1-A1 analyzed by flow cytometry, as shown in c. △MFI was determined by evaluating the change in mean fluorescence intensity of Calcein-AM-loaded iRBCs (Hoechst 33,342-positive subset, indicated with PB450A), after incubation with 100 μM DIP (N = 5, Mann Whitney test, *P < 0.05). e Representative images of the control PyDMT1-HA and Pydmt1-A1 parasite-infected RBCs after Calcein-AM treatment. Parasite-infected RBCs were incubated with Calcein-AM and DIP 1 h (the second row) or without (the first row). After three times washing, the iRBCs were transferred to the plate pretreated with poly-lysine, and imaged by Olympus FV3000 laser scanning confocal microscope. Quantitative data shown in f. f The LIPs of PyDMT1-HA and Pydmt1-A1 analyzed by confocal microscopy as shown in e. △MFI was determined by evaluating the change in mean fluorescence intensity of Calcein-AM in the region of parasite, after incubation with 100 μM DIP (N = 15 from three independent experiments, t-test, *P < 0.05.). g The schizont maturation of Pydmt1-A1 rescued with 100 μM Zn²⁺ (N = 80 parasites from three independent experiments. One-way ANOVA and Tukey’s multiple comparison test. ****P < 0.0001). h The schizont maturation of Pydmt1-A1 supplemented with 100 μM Cu²⁺ or Mn.²⁺ (N = 80 parasites from three independent experiments. One-way ANOVA and Tukey’s multiple comparison test. n.s. P > 0.05)