Extended Data Fig. 4: Chemical and genetic targeting of PKG and CDPK4.

(A) The number of parasites per infected RBC over time with PKG knockdown, assessed by flow cytometry. A single representative experiment is shown of three biological repeats. (B) The number of WT or PKG-DD-glmS parasites, with knockdown (no Shld1) or without knockdown (500 nM Shld1) induced for 24 h prior to initiating the experiment, that egress when treated with varying concentrations of 8-Br-cGMP. Each point is the mean of a biological replicate performed in technical triplicate. n = 3. (C) Sanger sequencing of pkg in the uncloned parasite population after transfection (T651Q modification). Positions 1-7 are the alternative bases in the homology repair template where editing is expected. (D) Alignment of the CDPKs and PKG surrounding the gatekeeper residue (boxed amino acid). (E) IC₅₀ of MMV030084 for proliferation over 72 h. Each point represents a biological replicate performed in technical triplicate. The mean ± SD is shown. n = 3. Statistical analysis shown is one-way ANOVA with Dunnett correction for multiple comparisons (two-sided). The ‘+’ condition was 500 nM Shld and the ‘-‘ condition was 250 nM Shld. (F) A representative flow-cytometry gating strategy to determine the percent of infected RBCs.