Fig 15. Inhibition correlates with cellular PmV levels.

(A) Western blot analysis of cellular levels of PmV in the parental line, 3D7, and clonal parasites generated by genetic modifications. Immuno-detection of expressed PmV (upper panel) and BiP (lower panel), used as a loading control on the same SDS-PAGE gel, are shown. DC6 clone expresses PmV-GFP chimera under regulation of the native promoter [15]; G6 clone, the PmV-GFP-DD chimera under regulation of the native promoter (this paper) and #3 clone (episomally expressing PmV-GFP chimera under Hsp86-5', a strong and constitutive promoter) [15]. G6 clone is maintained in absence of Shield-1. (B) Growth inhibition curves of parasites expressing different levels of PmV: 3D7 (blue dots and line), DC6 (green dots and line), G6 (PmV knock-down) (red dots and line) and #3 (Overexpressing PmV) (purple dots and line) were analysed in parallel. Growth inhibition was measured in triplicates. Dots represent actual data, lines the sigmoidal fitting obtained by non-linear regression analysis. (C) Shifts of inhibition curves detected by comparison of parasites expressing different levels of PmV. The sensitivities to Compound 29 (bars with solid fill) and Chloroquine (CQ—bars with meshed fill) of 3D7, DC6, G6 (PmV knock-down) and #3 (Overexpressing PmV) were analysed in parallel. CQ sensitivity for #3 is was not determined. Comparisons of IC₂₀, IC₅₀ and IC₈₀ values are shown in Table 1 and derived from curves shown in B. 3D7 and DC6 cultures expressed similar levels of Plasmepsin V; while G6 in the absence of Shield-1 showed decreased, and #3 showed augmented, levels of the cellular enzyme concentration. No significant shift were detected with CQ. Error bars show the standard deviation of the data.