Figure 2. Kelch13 WT and Kelch13 mutant parasites differentially regulate the UPR.

(a) UPR activation was assessed in WT and R539T parasites under the depicted conditions. Red arrows indicate addition of drug and red lines indicate drug treatment. The blue triangle indicates time of drug washout and the blue line indicates time after washout. (b) WT and R539T parasites were synchronized to 0–3 hpi rings and treated with DMSO or 700 nM DHA for 3 h, then lysates were subject to Western blot and immunoblotted with antibodies against p-eIF2α and eIF2α. Shown is a representative blot from three independent experiments (see Supplementary Fig. 2a for replicates). (c) Densitometry analyses was performed using Image J and UPR activation determined as described in Fig.1. (d) WT and R539T parasites were synchronized to 0–3 hpi rings and treated with DMSO or 700 nM DHA for 3 h. Then drug was washed off and parasites were harvested at the indicated times to monitor UPR resolution. Western blot was performed as described in (b). Representative blot shown from three independent experiments (see Supplementary Fig. 2b for replicates). (e) Densitometry and determination of UPR activation performed as described in (c). (f) Rate of de-phosphorylation of eIF2α following drug removal was calculated over time and the mean negative slope ± S.E.M. was plotted. (g) WT and R539T parasites were synchronized to 26–30 hpi trophozoites, then treated with DMSO or 50 nM DHA for the indicated times. Western blot was performed as described in (b). Representative blot shown from four independent experiments (see Supplementary Fig. 2f for replicates). (h) Densitometry and determination of UPR activation performed as described in (c). Bar graphs indicate mean normalized integrated density ± S.E.M. from at least three independent experiments. Statistical significance was examined for the indicated comparisons using a two-tailed paired t-test. *p < 0.05; n.s. = not significant.