FIG 6.

(A) IFA imaging of DMSO- and RAP-treated CDC50C-HA:loxP trophozoites fixed at 36 h postinvasion indicates no defect in export of skeleton binding protein (SBP). Right, Western blot showing absence of CDC50C-HA in RAP-treated trophozoites. Scale bar, 2 μm. (B) Quantification of SBP puncta in DMSO- and RAP-treated CDC50C-HA:loxP trophozoites. Sixty-six parasites were counted from two independent experiments for SBP puncta in ImageJ. Mean values are plotted. Error bars, SD; n.s, not significant, Student's t test. (C) CDC50 null parasites produce smaller hemozoin crystals. Thin blood films were made from tightly synchronized DMSO- and RAP-treated CDC50C-HA:loxP parasites at 36 h postinvasion. Inset, length of the hemozoin crystal (measurement 1) and parasite (measurement 2) were performed in ImageJ on imaged Giemsa-stained smears of DMSO- and RAP-treated CDC50C-HA:loxP trophozoites. In total, 60 control or RAP-treated parasites were measured from three independent experiments. Mean values are plotted. Error bars, SD; *, P < 0.05, Student's t test. (D) Spectrophometric quantification of the effects of CDC50C ablation on parasite hemozoin content. Highly synchronized ring stage CDC50C-HA:loxP cultures were treated with DMSO (control) or RAP. Cultures were harvested at 36 h postinvasion, and hemozoin was purified using established methods (45) and then quantified by absorbance at 410 nm. Means are plotted for three independent experiments. Error bars, SD; n.s, not significant; *, P < 0.05, Student's t test. (E) Western blot analysis of the effects of CDC50C ablation on hemoglobin content. Highly synchronized ring stage CDC50C-HA:loxP cultures were treated with DMSO (control) or RAP. Cultures were then harvested at 36 h postinvasion, and parasites were released using saponin. Parasite extracts were probed for the presence of CDC50C by HA staining. Hemoglobin content was probed alongside GAPDH as a loading control. Data are representative of three independent experiments. (F) Effects of CDC50C ablation on E64-mediated food vacuole bloating. Tightly synchronized DMSO- or RAP-treated CDC50C-HA:loxP parasites were treated with 33 μM E64 at 24 h postinvasion and left to develop for a further 8 h, after which they were stained with 4.5 μg/mL dihydroethidium to detect the food vacuole and imaged. A minimum of 20 cells were counted per condition and scored for bloated or nonbloated food vacuoles. An inset shows representative images of bloated and nonbloated parasites; scale bar, 2 μm. Mean data are plotted for three independent experiments. Error bars, SD; *, P < 0.05, Student's t test for the comparison of nonbloated parasites between DMSO and RAP treatments.