Extended Data Figure 6: Effect of AMPK agonists.

a, Dose-dependent effect of AMPK activators (salicylate and A769662) on P. berghei ANKA that express luciferase (under ama1 schizont-specific promoter). Parasites were cultured for 24 h with increasing concentrations of the compounds (media supplemented with 20% FBS and 5 mM glucose). Analysis of schizont development was performed by measuring luminescence. Values are mean ± s.e.m. (salicylate, n = 5; A769662, n = 6). EC50 values determined by using GraphPad Prism nonlinear regression variable slope (normalized) analysis. The calculated EC50 values are as follows: salicylate, 2.4 ± 0.9 mM; A769662, 256.5 ± 60.6 μM. b, Dose-dependent effect of A769662 on P. falciparum, P. berghei ANKA wild-type, Δkin and complemented parasites analysed by microscopy after Giemsa staining (Mann–Whitney test). Box plots show the data for the following number of schizonts (vehicle, A769662 62.5 and 125 μM): P. falciparum, 59, 18, 48; wild-type P. berghei, 28, 68, 61; Δkin, 44, 47, 40; Δkin+kin, 34, 57, 37. c, Dose-dependent effect of A769662 and salicylate treatments on P. falciparum for two developmental cycles. Synchronized cultures were set at 0.1% initial parasitaemia (rings) and analysed at 48 h and 96 h by flow cytometry after SYBR Green labelling of parasite DNA. A new generation of rings was observed at 48 and 96 h in the treated conditions, suggesting no growth delay. Data (mean ± s.e.m.) was normalized to the untreated control on each experiment at 48 h or 96 h. The EC50 values (determined as in a) are as follows: 133.1 ± 3.4 μM at 48 h (n = 6) and 70.1 ± 18.9 μM at 96 h (n = 5) for A769662; 2.2 ± 0.2 mM at 48 h (n = 6) and 1.25 ± 0.2 mM at 96 h (n = 6) for salicylate. d, qPCR analysis of P. falciparum parasites treated with salicylate for 72 h (n = 2 per condition). Data normalized to the untreated control. The genes analysed correspond to the P. berghei homologues experimentally validated in Extended Data Fig. 3. Gene IDs are shown in the figure without the ‘PF3D7_’ prefix. e, Dose-dependent effect of salicylate on other P. berghei ANKA kinase mutants. Box plot of parasites treated and analysed as in b (Mann–Whitney test). The number of schizonts analysed for vehicle, 0.6 mM and 1.25 mM are as follows: Δnek2, 43, 33, 37; Δcdpk3, 35, 44, 30. f, g, Salicylate effect in vivo. Mice were treated daily with 250 mg kg−1 salicylate (sal) or 0.9% NaCl (vehicle, veh) starting at day 1 after infection. Parasitaemia (mean ± s.e.m.; two-way ANOVA test) and survival (log-rank Mantel–Cox test) of C57BL/6 mice infected by i.p. injection of 1 × 106 wild-type (veh n = 15; sal n = 16), Δkin (veh n = 8; sal n = 8) and complemented Δkin+kin iRBCs (veh n = 10; sal n = 10).