Fig. 4: Lack of GRA12 results in IFNγ-mediated host cell death by necrosis.

a Time course of PWD/PhJ BMDMs cell death quantified via Propidium Iodide uptake. The graph shows the median value ± SEM, N = 4. Cells were pre-stimulated for 24 h with IFNγ (continuous line) or left untreated as control (dashed line) and then infected with VAND ∆UPRT, ∆GRA12, ∆GRA12::GRA12-HA or left uninfected. The number of dead cells is expressed as a percentage of the total at each time point. b Cell death quantification at 9 hpi of IFNγ-treated PWD/PhJ BMDMs infected with ∆UPRT, ∆GRA12, ∆GRA12::GRA12-HA in the PRU, RH and VAND strains, or left uninfected as control. The bar graph shows the median value ± SD, N > = 4. Significance was tested using the Tukey’s multiple comparisons test for the RH and VAND strains, and with a paired t test for the PRU strains. c Western blot analysis of the supernatant and cell lysate of C57BL/6J BMDMs pre-treated with IFNγ and infected for 8 h with RH ∆UPRT, ∆GRA12, ∆GRA12::GRA12-HA, or left untreated as control. d Live cell imaging of IFNγ-treated C57BL/6J BMDMs infected with RH ∆GRA12 parasites. Time post infection reported as hh:mm in the bottom right corner. Filled arrowheads indicate dying cells and an egressed parasite. Complete video is in Supplementary Movie 1. e Fluorescence microscopy detection of RH ∆UPRT parasites after 48 h growth in HFFs treated or not with 1 µM ML10. Scale bar represents 20 µm. f Time course of C57BL/6J BMDMs cell death quantified via Propidium Iodide uptake. Cells were pre-stimulated for 24 h with IFNγ, and infected with RH ∆UPRT, ∆GRA12, ∆GRA12::GRA12-HA before treatment with 1 µM ML10 (triangle symbol), or left untreated as control (continuous line). The number of dead cells is expressed as a percentage of the total at each time point. g Cell death quantification at 9 hpi of the ML10 treatment experiment. The bar graph shows the median value ± SD, N > = 4. Symbol shapes indicate biological repeats. Significance was tested using the One-way Anova test with the Benjamini, Krieger and Yekutieli FDR correction. Source data are provided as a Source Data file.