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. 2021 Jun 18;12:3788. doi: 10.1038/s41467-021-24083-y

Fig. 5. RON13 is critical for invasion.

Fig. 5

a Plaque assays of different parasite strains (±ATc) showing that depletion of RON13 impairs the parasite lytic cycle (no plaque), a phenotype that is fully rescued by complementation with an active RON13 kinase. Image representative of three biologically independent experiments. b Quantification of plaque assays for RH, RON13-KD, and complemented RON13-KD/ron13wt or RON13-KD/ron13dk parasites (±ATc). Data are presented as box and whiskers plot (median with min to max, n = 3 biologically independent experiments). Statistical significance was assessed by a two-way ANOVA significance with Tukey’s multiple comparison. c Invasion assay (+ATc) showing a strong invasion defect when RON13 kinase is absent or inactive P value were determined by a one-way ANOVA significance with Tukey’s multiple comparison. (Mean ± SD; n = 3 biologically independent experiments). d IFA showing a representative field of the rhoptry secretion test using phosphor-STAT6 as a readout with fibroblast nuclei stained in DAPI (magenta) and ROP16-injected cells stained with anti-phospho-STAT6 (STAT6-P) antibody (green, asterisks). Scale bar = 25 μm. Image representative of three biologically independent experiments. e Phospho-STAT6 assays assessing the ability of the parasite to secrete the rhoptry protein ROP16 into the host cell that phosphorylates host STAT6 in the nucleus. Data are presented as box and whiskers plot (median with min to max, n = 3 biologically independent experiments). Statistical significance was assessed by a two-way ANOVA significance with Tukey’s multiple comparison. f Representative IFA of the e-vacuole assay to assess rhoptry secretion when invasion is blocked by cytochalasin D. A parasite (p) secreting e-vacuoles ROP1+ (asterisks) is depicted. Parasite DNA is visualize using DAPI (blue). Scale bar = 5 µm. Image representative of three biologically independent experiments. g E-vacuole assays assessing the ability of the parasites to secrete the rhoptry protein ROP1 into the host cell (mean ± SD; n = 3 biologically independent experiments). Statistical significance was assessed by a one-way ANOVA significance with Tukey’s multiple comparison. h Microneme secretion of extracellular parasites stimulated with 2% ethanol to assess the release of MIC2 in culture supernatant. pMIC2 processed MIC2, ESA excreted–secreted antigens. GRA1 is used as a control for constitutive secretion from dense granules. Samples derived from the same experiment and gels were processed in parallel. Image representative of three biologically independent experiments. i Kinetic assay representing the cell index of HFF infected with different parasite strains. (Mean ± SD; n = 3 biologically independent experiments). j Invasion assay showing that RON13-KD and RON4-KO parasites are defective in invasion. P values were determined by a one-way ANOVA significance with Tukey’s multiple comparison (mean ± SD; n = 3 biologically independent experiments). Statistical significance was assessed by a one-way ANOVA significance with Tukey’s multiple comparison. k Virulence of different strains in mice. Surviving mice at 84 days post infection were challenged with RH parasites (gray shaded). (n = 5 biologically independent animals). P values were determined by a Mantel–Cox test. Source data are provided as a Source data file.