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. 2020 May 15;16(5):e1008572. doi: 10.1371/journal.ppat.1008572

Fig 4. GRA24 controls MyD88-independent p38 MAPK-dependent IL-12 production.

Fig 4

(A) MyD88+/+ and MyD88-/- BMDM were infected with cps1-1 or cps1-1:Δgra24 tachyzoites at the indicated parasite to cell ratios. Cultures were initiated with (+) and without (-) exogenous uracil and supernatants were collected 48 hr after infection. C, control non-infected cells. (B) MyD88-/- BMDM were inoculated with cps1-1, cps1-1:Δgra24, cps1-1:gra24C1 or cps1-1:gra24C2 tachyzoites at a 2:1 parasite to cell ratio. Supernatants were collected 48 hr post infection for IL-12 measurement. Production of IL-12 was significantly reduced in cells infected with cps1-1:Δgra24 in pairwise comparisons with cps1-1 and the two complementation mutants. (C) cps1-1 or cps1-1:Δgra24 tachyzoites were inoculated onto MyD88+/+ and MyD88-/- BMDM at a ratio of 2:1. Cultures were briefly centrifuged to initiate parasite and cell contact, then lysates were prepared for Western blot assay at the indicated time points after infection (hr). (D) WT BMDM were infected with cps1-1 parasites in the presence and absence of uracil, then lysates were prepared for Western blot analysis as in (C). (E) WT BMDM were infected with cps1-1 tachyzoites in the presence and absence of p38 MAPK inhibitor SB202190 (10 μM). Supernatants were collected 36 hr after infection for cytokine ELISA. The inhibitor was added either 1 hr prior to infection (“Early add”) or 10 hr after infection (“Late add”). Data shown are the means ± SD of cells cultured in triplicate. Statistical significance was evaluated using Student’s t test (* p < 0.05, *** p < 0.001). These collective experiments were repeated with the similar result two times.