FIG 3.

Host cell cycle progression is not required for robust and complete liver stage Plasmodium development in vitro. (A) Schematic illustrates experimental setup for aphidicolin block. At 48 h after aphidicolin washout, HepG2 cells were propidium iodide labeled and profiled by flow cytometry to determine the percentage of single cells in the G₁, S, and G₂-M phases of the cell cycle. Data (means ± standard deviations) shown are from one representative experiment with technical quadruplicate wells with at least 10,000 cells processed per well. (B) Aphidicolin block was performed as described for panel A; 48 h after aphidicolin washout and GFP-expressing P. berghei sporozoite infection, the geometric mean amounts of GFP (a quantitative readout of parasite growth) from the control and aphidicolin-treated cells were determined by flow cytometry. Data represent the means and standard deviations of the results of 3 independent experiments. (C) Aphidicolin block (AphBlock) was performed as described for panel A, but infection was allowed to proceed for 65 h before cells were fixed and immunolabeled with MSP-1, shown in red in the left-most panel, anti-GFP antibody (PbGFP, GFP-expressing P. berghei), in green, and DAPI (nuclei), in blue. The white square inset in the merged image of mature hepatic merozoites delineates the area presented at higher magnification in separate grayscale images for each of the three labels. The images are projections of confocal slices through the parasite (scale bars = 5 μm). (D) Two hundred microliters (of 500 μl total) of merosome-containing medium from aphidicolin-blocked or control HepG2 cells 65 h postinfection with GFP-expressing P. berghei sporozoites was injected i.v. into C57BL/6 mice, and blood stage infection was subsequently monitored by flow cytometry from day 2 through day 5 after merosome injection. Each point represents the mean parasitemia of 5 animals; error bars represent standard deviations.