Figure 4. 19S proteasome mutants display increased susceptibility to DHA.

(a) Diagram of 26S proteasome showing placement of Rpt5, Rpn6, and Rpn4 within the multisubunit complex. (b-d) WT, Rpt4, Rpn6, C580Y, and Rpt5 parasites were tightly synchronized to 0–3 hpi rings, then exposed to a range of DHA concentrations starting at 1400 nM DHA for 3 h. DHA was washed off, and parasitemia was assessed 66 h later. (b) % survival was calculated by dividing the parasitemia of parasites treated with 700 nM DHA by the parasitemia of mock-treated parasites. (c) ring-stage IC₅₀ and (d) ring-stage IC₉₀ values were determined using non-linear regression analysis. (e) Parasites were tightly synchronized to 26–30 hpi trophozoites, then exposed to a range of DHA concentrations for 3 h. DHA was washed off, parasitemia assessed 66 h later, and trophozoite-stage IC₅₀ values determined as above. (f) Dose response assays with DHA were conducted on asynchronous parasites, parasitemia assessed 72 h later, and IC₅₀ values determined as above. Bar graphs show mean IC₅₀ or IC₉₀ values ± S.E.M., determined from at least four independent experiments. Statistical significance was examined for each proteasome mutant against the cognate parental strain using a Mann-Whitney U test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. = not significant.