Fig. 3

Dihydroartemisinin (DHA) alters the PfAP2-G expression profile and inhibits parasitic development. a Schematic illustration of the experimental setup: the transcriptional profiles of five GRG markers were built for synchronized NF54 trophozoite-stage parasites treated over the course of one 48-h cycle after a short (3-h) pulse with DHA (5 or 10 nM) or 0.1% DMSO (solvent control) or non-treated (NT) control. The flask and arrow represent treatment induction starting at the first time point (0 hpt). RNA was extracted by the robotic unit at 4, 12, 25, 30 and 48 hpt for RT-qPCR expression analysis. The predominant stage in each time span is also presented in the illustration for comparison with the transcriptional profiles. b–f Temporal transcriptional profiles of the early gametocytogenesis markers PfAP2-G (b), gexp05 (c), Pfg14.748 (d), Pfs16 (e) and sbp1 (f, a ring-stage marker). g Transcript levels of Pf GRGs: PfAP2-G, gexp05, Pfg14.744, Pfg14.748, Pfs25 and sbp1, 24 h post-DHA induction under 2 mM choline treatment. h Growth assay for NF54 Pf-iRBCs post-treatment with DHA (5 nM or 10 nM) for 3 h (trophozoite stage), monitored by flow cytometry. The parasitemia was quantified every 24 h over a 72-h period. The respective volumes of DMSO (0.05% and 0.1%) were used as solvent controls, and non-treated (NT) parasites were used as negative control. The flow cytometry gating strategy for a representative example can be observed in Additional file 14: Fig. S11. In Panels b–f and h, data represent the mean of three independent biological repeats (n = 3) with three technical repeats each, while in Panel g, data represent the mean of five independent biological repeats (n = 5) for all the genes except for sbp1 (4 biological repeats, n = 4). Mean transcript levels were calculated using the relative standard curve method and normalized to the transcript levels of uce in Panels b–f. Error bars represent SD. Two-way ANOVA with post-hoc tests were run using estimated marginal means with the R package 'emmeans' for Panels b–f and h. An unpaired t-test was performed between the DHA-treated and non-treated parasites for each gene individually in Panel g. *p < 0.05, **p ≤ 0.01 and ****p ≤ 0.0001