Effect of PfPK2 depletion on parasite division and reversal of phenotypic defects upon episomal expression of PfPK2.

A. Western blot was performed on lysates prepared from cHA-PfPK2-loxP parasites, which episomally express HA-PfPK2 after DMSO or RAP treatment using anti-GFP, anti-HA or anti-BiP (loading control) antibodies. There was no loss of HA-PfPK2 whereas GFP-PfPK2 was depleted upon RAP-treatment. B. PfPK2-loxP parasites were treated with either DMSO or RAP at ring stages. After ~40–44 h.p.i parasites were treated with E64, and mature segmentor stage parasites were traced. Giemsa-stained blood smears were prepared and number of merozoites per schizont were counted and % schizonts possessing indicated number of merozoites was determined (SEM ± SE, n = 2, ANOVA, P>0.05; ns -not significant). C. PfPK2-loxP or cHA-PfPK2-loxP parasites were treated with DMSO or RAP and the number of abnormal/pyknotic parasites upon RAP addition were counted after 72 hpi (as described for Fig 2G). A significant number of abnormal parasites were observed in PfPK2-loxP upon RAP addition whereas the change observed in cHA-PfPK2-loxP parasites was not significant (SEM ± SE, n = 3, ANOVA, ** PD. Invasion assays were performed as described in Fig 3A, for indicated lines in the presence or absence of treatment of schizonts with DMSO or RAP and the number of rings were counted after 12h, which reflected invasion. Fold change in invasion upon RAP treatment was determined (SEM ± SE, n = 3, ANOVA, *** P0.05; ns -not significant. (PDF)