In vitro selection of HU-resistant Pf3D7 strain. (A) Diagram of selection schemes. Stepwise selections: 3 independent parasite cultures were exposed to stepwise increasing concentrations of HU (131.5-854.65 μM) as indicated. (B) A representative dose-response curves of HU against parental strain (3D7wt) and bulk-selected HU-resistant parasite populations at different time points of the selection (3D7HU15, 3D7HU30, and 3D7HU65). (C) Shift in EC₅₀ of bulk-selected HU–exposed parasite populations at different time points of the selection (3D7HU15, 3D7HU30, and 3D7HU65). (D) Shift in EC₅₀ of the bulk-selected 3D7HU65 parasite populations and 4 clones with higher (2F10-1E9, 2F10-2E10) or lower (1F5-2F4 and 1F5-2C9) EC₅₀. (E) Dose-response curves of clones 2F10-2E10 (i) and 1F5-2F4 (ii) and parental Pf3D7 (3D7wt). (F) Shift in EC₅₀ of clones 2F10-2E10 (i) and 1F5-2F4 (ii). (G) Growth response of clones 2F10-2E10 (i) and 1F5-2F4 (ii) maintained in media without HU (CM) or with HU at 6.5× EC₅₀ (CM+HU65). Parental Pf3D7 strain (3D7wt) is also shown. In panels C-D, F, the horizontal bar represents the median and the vertical bar the interquartile range. For the mean comparisons, 1-way ANOVA with a Tukey post hoc analysis was used in panels C-D, and the Mann-Whitney test was used in panel F.