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. 2020 Oct 16;11:5258. doi: 10.1038/s41467-020-18991-8

Fig. 2. Validation of candidate genes that determine parasite fitness in IFNγ-stimulated murine BMDMs.

Fig. 2

a–f Murine BMDMs prestimulated with IFNγ (1 or 5 ng/mL) or left unstimulated for 24 h were infected with luciferase-expressing wild-type (WT) parasites or with parasites in which TGGT1_269620 (a, n = 6), GRA45 (b, n = 5), TGGT1_232670 (c, n = 5), TGGT1_263560 (d, n = 5), GRA22 (e, n = 4), or TGGT1_269950 (f, n = 5) was knocked out (MOI of 0.25). Parasite growth for each strain was measured by luciferase assay at 24 h p.i. Parasite growth in IFNγ-activated BMDMs is expressed relative to growth in naïve BMDMs. Data are displayed as mean ± SEM with independent experiments indicated by the same color dots. The significant difference between WT and knockout was analyzed with two-tailed paired t-test. g, h Growth competition assay between GFP-positive WT parasites and GFP-negative g Δgra22 or h Δrom1 parasites was performed in murine BMDMs prestimulated with 1 ng/mL IFNγ or left unstimulated for three passages. The percentage of Δgra22 or Δrom1 was determined at the start of the competition and after three passages by plaque assay measuring the GFP-negative plaques vs. total plaques. Data are displayed as mean ± SEM with independent experiments (n = 3) indicated by the same color dots. The significant difference between the ratio of WT vs. knockout before and after competition was analyzed with two-tailed paired t-test.