Overexpression of PfPKAc in NF54/PKAc cOE M1 parasites is lethal but survivor populations tolerant to PfPKAc OE can be selected. (A) Expression of PfPKAc-GFP in NF54/PKAc cOE M1 parasites under OE-inducing (–GlcN) and control conditions (+GlcN) as assessed by live cell fluorescence imaging and western blot analysis. Synchronous parasites (0 to 8 hpi) were split (±GlcN) 40 hours before sample collection. Representative fluorescence images are shown. Parasite DNA was stained with Hoechst. Scale bar = 5 μm. In 2 clones (NF54/PKAc cOE M1 and M2), correct integration of the inducible PfPKAc-GFP OE expression cassette and absence of WT parasites was confirmed by PCR on gDNA. Live cell fluorescence imaging and western blot analysis confirmed the efficient induction of PfPKAc-GFP OE upon GlcN removal in both clones (A. Interestingly, PfPKAc-GFP OE (–GlcN) resulted in a complete block in parasite development half way through the IDC. (E) Expression of PfPKAc-GFP in NF54/PKAc cOE S1 survivor parasites under OE-inducing (–GlcN) and control conditions (+GlcN) as assessed by live cell fluorescence imaging and western blot analysis. Parasites were cultured and samples prepared as described in panel A. Scale bar = 5 μm.

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