BODIPY-ceramide and FM4-64 staining of live infected erythrocytes in the presence and absence of anti-P2 mAb E2G12.

<p>(<b>A–C</b>) Control, anti-P2-mAb E2G12 and anti-β actin antibodies were added to synchronized cultures of <i>P. falciparum</i> at 12 hrs post merozoite invasion. Cells were harvested from 24 hrs to 36 hrs at various time points, washed, incubated with BODIPY-ceramide for 15 min at 37°C, and the structure of TVN <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002858#ppat.1002858-Straschil1" target="_blank">[41]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002858#ppat.1002858-Tewari1" target="_blank">[42]</a> was recorded in IEs. <b>D.</b> Quantification of BODIPY ceramide intensity in the infected RBC cytosol averaged over at least 15 infected red cells at various time points under control (no antibody), and in the presence of E2G12 and anti-β actin antibodies as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002858#s4" target="_blank">Materials and Methods</a>. <b>E–H.</b> Infected erythrocytes were treated with FM4-64 for 30 mins to measure the lipid import <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002858#ppat.1002858-Tewari1" target="_blank">[42]</a>; <b>E.</b> Flow cytometric analysis depicting mean fluorescence intensity (MFI) of FM4-64 uptake in infected RBCs at various time points; with no antibody (Ab), in the presence of anti-P2 mAb E2G12 and after removal of E2G12 (washed) at 36 hrs PMI. Significance (<i>*P</i><0.05 and <i>**P</i><0.01) of each data point was calculated with respect to the immediate previous data point. (<b>F–H</b>) Confocal microscopy of FM4-64 uptake under control (no antibody); with anti-P2 mAb E2G12 and anti-β actin treated cells at various stages of development. In all images nuclei were stained with DAPI. Scale bar indicates 2 µm.