Fig. 1: The transgenic strains were resistant to DHA.

The 20 µg of parasite protein samples were separated and transferred, which were then immunoblotted with anti-GFP antibody (A) and anti-mCherry antibody (B). A An anti-GFP antibody detected a major protein band of 165 kDa, which agreed with the predicted size of PfKelch13 fused with two FK506-binding protein (FKBP) domains and GFP. B An anti-mCherry antibody detected a protein band of 130 kDa, which agreed with the predicted size of VPS34 fused with mCherry. C Confocal images of untagged 3D7 (control), 3D7-PfKelch13^WT, 3D7-PfKelch13^C580Y, and 3D7^VPS34. Tagged exogenous 3D7-PfKelch13^WT and 3D7-PfKelch13^C580Y are evident in the GFP panel, and VPS34 is evident in the red panel, while the nuclei are stained with DAPI, the parasites were visualized via brightfield microscopy, and images were merged for reference. D In vitro antimalarial activity of DHA against 3D7-PfKelch13^WT, 3D7-PfKelch13^C580Y, and 3D7^VPS34. The IC₅₀ values were calculated by GraphPad Prism 9.0 software (Analyse-Nonlinear regression curve fit-[Inhibitor] vs. normalized response—variable slope). The definitions of the maximum and minimum response values were 100% and 0%, respectively for the nonlinear regression curve fit. E The PI3P levels and the RSA values of 3D7 (control), 3D7-PfKelch13^WT, 3D7-PfKelch13^C580Y, and 3D7^VPS34. For the PI3P level measurement, the parasites were not treated with any drugs. When the parasites reached the trophozoite stage, the red blood cells were lysed with saponin, and the lipids within parasites were extracted for PI3P detection. For the RSA value measurement, tightly synchronized early-ring parasites (0–3 h post-invasion, 2% hematocrit and 1% parasitemia) were exposed to 700 nM DHA combined (0.1% DMSO as control) for 6 h, washed thrice with Albumax-free RPMI and returned to culture for 66 h. Parasite survival rates (%RSAs) were determined by comparing the number of viable parasites between the drug-treated and untreated control and are expressed as percentages. Two-tailed unpaired Student’s t-test was employed. The mean (s.d.) are based on three replicates (the triplicate data points are shown). PI3P: **p < 0.01, ***p ≤ 0.001; RSA: ^###p ≤ 0.001.