Fig. 7: The combination of DHA and idelalisib led to increased accumulation of ubiquitinated proteins (A, B) and increased eIF2α phosphorylation (C, D).

In addition, idelalisib upregulated the expression of genes in the UPR pathway in 3D7-PfKelch13^WT (E) but downregulated them in 3D7-PfKelch13^C580Y (F). Tightly synchronized ring-stage P. falciparum 3D7 cultures were exposed to 0.1% DMSO (control), DHA at the IC₅₀, idelalisib at the IC₅₀ or the combination of DHA and idelalisib (each at the corresponding IC₅₀) for 24 h until the parasites reached the late-trophozoite stage. After incubation, the erythrocytes were lysed with saponin and the parasite pellets were washed three times with PBS. Each treatment group had six replicates, with three replicates used for RNA extraction and another three replicates used for protein extraction. For Western blot analysis, 20 µg of parasite protein samples was separated and transferred which was then immunoblotted with anti-ubiquitin (A, B), anti-eIF2α (C, D) or phospho-eIF2α (C, D) antibodies. For RT-qPCR, the RNA was extracted. The expression levels were normalized to the level of the housekeeping gene seryl-tRNA synthetase. Relative expression (fold change) was calculated from the expression levels of the control of 3D7-PfKelch13^WT. Values greater than 1 (dashed line) indicate upregulation. One-way ANOVA and LSD-T post hoc test comparisons between treatment groups were employed. The means (s.d.) are based on three replicates (the triplicate data points are shown). p < 0.05, **p < 0.01.