Fig. 2. Treatment with the MC2646 compound restores host cell autophagy.

a Ultrastructural analysis by electron microscopy revealed classical autophagy-related organelle structures in uninfected BL3 cells, but a reduction of these structures in infected TBL3 cells. Treatment with MC2646 restored increased autophagosome in both cells. The electron micrographs are shown at a magnification of 2900x (left) and the highlighted blue box at 9300x (right). The white arrowheads highlight the autophagosome structures in BL3 cells and both cell types treated with MC2646. The red arrow highlights the annulate lamellae visible in infected cells. b Gene Signature Enrichment Analysis (GSEA) where color scale represents Normalized Enrichment Score (NES) and circle size represents p-value. Gene Ontology Biological Process (GOBP) genesets related to autophagy were used to compare the four RNA-Seq datasets: uninfected BL3 & infected TBL3, treated with Buparvaquone or MC2646, compared to the respective untreated dataset. Crosses indicate enrichment scores that are not statistically significant (FDR q-value > 0.25). The GSEA was evaluated with a Weighted Kolmogorov-Smirnov Test. c Analysis of LC3B protein in infected TBL3 and uninfected BL3 cells with or without MC2646 treatment. Western blots profiles show LC3-I and LC3-II forms which are restored in TBL3 cells after MC2646 treatment (4 µM, 24 h). The quantification of the ratio between LC3-II:LC3-I is indicated below. Actin was used as a loading control. d Infected TBL3 cells were untreated (Ctrl) or incubated with MC2646 (4 µM for 24 h) and LC3B-puncta were monitored by immunofluorescence (green). Host and parasite nuclei are indicated by DAPI staining. Leica microscope x63 magnification; the scale bar corresponds to 15 µm. e Autophagy induced by low-nutrient media reduced parasite survival. Infected TBL3 cells were incubated in EBSS media (4 h) and parasite load (determined by number of nuclei per host cell) was monitored. The addition of BafilomycinA1 (BafA1), blocks autophagic flux and rescued parasite survival. At least 50 host cells were counted per condition. Results are significant under Kruskal-Wallis followed by a Dunn’s multiple comparison test *p < 0.0191; ****p < 0.0001. f Quantification of LC3B puncta (same conditions as Fig. 2e). In all experiments cells were incubated with 50 ng/ml Buparvaquone or 4 µM MC2646 for 24 h. Results are representative of 3 independent experiments. Statistical analysis Dunnett’s multiple comparison test. *p < 0.1, ****p < 0.0001.