The effect of PfPI3K inhibition on hemoglobin trafficking. (A) Parasites were treated with DMSO or wortmannin. Saponin treatment was used to release excess nonparasitic hemoglobin, followed by attachment of parasites onto poly-lysine-coated glass cover slips. After fixation in paraformaldehyde/glutaraldehyde and Triton X-100 permeabilization, IFA was performed using anti-hemoglobin antibody, and 4,6-diamidino-2-phenylindole was used to stain the parasite nucleus. A single erythrocyte-free parasite can be seen in each phase-contrast image (PC) with its food vacuole. The thick arrow represents the collection of hemozoin crystals, which indicates the position of the food vacuole. Corresponding hemoglobin immunofluorescence images (Hb) contain punctate vesicle-like structures indicated by the thin arrow. (B) Average transport vesicle counts per parasite were compared between wortmannin and DMSO control. Error bars represent SEM. (C) Transmission electron micrographs of DMSO and 10μM wortmannin-treated trophozoite stage malaria parasite. Each micrograph shows a single parasite located inside a host erythrocyte. The cytoplasm of the host cell is darkly stained because of the preponderance of electron-dense hemoglobin. Labeled structures inside the parasites are the parasite nucleus (N), food vacuole (Fv), and hemoglobin transport vesicles (V).