Fig. 4. Pharmacological treatments impact parasite differentiation.

a KDMi and KDACi treatment impacted merogony induction in infected cells. Quantification of cells at macroschizont (orange) or merogony (green) parasite stages after 8 days cultured at 41 °C treated with inhibitors, biologically independent experiments in triplicate, mean of the cells in schizont or merogony stage for each replicate. The right panels shows the expression levels of the parasite gene Tamr1 (merogony marker) after 8 days at 41 °C treated with inhibitors. Levels at 41 °C were compared to 37 °C schizont controls n = 3 biologically independent experiments represent qPCR values of TamR1 expression normalized by Hsp70 for three different induction of merogony treated or not. Error bars represent mean values ± SD. Statistical test Dunnett’s multiple comparison and two-sided test: ns = not statistically significant, p = 0.1195; ****p = 0.0001; **p = 0.0031. b Cluster IV genes are over-represented in differentially expressed genes in sporozoite-tomacroschizont (S2M down-regulated) and macroschizont-to-merozoite (M2M up-regulated) transitions. Dot chart of the representation factor and p-value of significant overlap between differentially expressed genes derived from a published microarray¹⁹ and our H3K18me clusters. Dot colour transparency is plotted as p-value and dot size represents the representation factor. The right panel shows Venn diagram indicating the numbers of genes overlapping between cluster IV, genes upregulated in the M2M transition and genes downregulated in the S2M transition. c Merogony induction increased the expression of the M2M13 gene (Cluster IV) that is upregulated during macroschizont to merozoite transition. The M2M13 gene was affected by the epigenetic inhibitors KDMi and KDACi. RT-qPCR analysis of M2M13 gene in infected macrophages, at 37 °C or at 41 °C for 8 days, after treatment with inhibitors, showed that the expression level of M2M13 was significantly reduced by KDMi treatment. Levels at 41 °C were compared to 37 °C schizont controls or untreated merogony (n = 3 biologically independent experiments, mRNA expression of M2M13 normalized to Hsp70, for three different experiments on schizont treated or not and for three inductions of merogony treated or not). Error bars represent mean values ± SD. Statistical test Dunnett’s multiple comparisons two-sided: ns = not statistically significant > 0.99; **p < 0.005; *p = 0.0312.