FIGURE 4.

Transfer of GzmB is mediated by GNLY in a stage-specific manner. Uninfected and nonsynchronized iRBC cultures were treated with Alexa Fluor 488–labeled GzmB (0.4 μM) in combination with PFN (0.25 hu) or GNLY (0.5 μM) for 20 min before fixation and assessment by confocal microscopy. Intraerythrocytic forms (early and late stages) are shown in (A) and Supplemental Fig. 1, and merozoites are presented in (C). Nuclei were stained with Hoechst. Scale bars, 2 μm in (A) and 5 μm in (C). (B) Over 100 cells per condition in four independent experiments were assessed for intracellular GzmB and quantified. Averages ±SEM of GzmB-488–containing cells in three independent experiments are shown. The p values of differences between groups, calculated by Student t test, are indicated. Representative visual fields used for the quantification are shown in Supplemental Fig. 1. (D) Unsynchronized iRBCs of high parasitemia were treated with PFN or GNLY for 5 min in presence of fluorescently labeled dextran and Hoechst and then assessed live by confocal microscopy. Late stages are indicated with white arrows, early stages are indicated with black arrows, and uninfected RBCs are indicated with a gray arrow. Scale bar, 10 μm. (E) Dextran uptake in late- and early-stage iRBCs upon GNLY or PFN treatment was quantified by counting 30 cells per condition in three independent experiments.