Fig. 2: AMPKγ is essential for the growth of Toxoplama tachyzoites.

a Depletion of TgAMPKγ in the AMPKγ-mAID strain by IAA treatment, as determined by IFA on intracellular parasites treated with or without IAA. The anti-HA antibody was used to probe the level of AMPKγ and TgALD was used as cytoplasmic marker. b Lack of parasite growth after TgAMPKγ depletion, as determined by plaque assays with or without IAA treatment for 7 days. c Relative sizes of plaques (expressed as pixel units when calculated from Adobe Photoshop) derived from b. More than 90 plaques were determined for each condition and the data are graphed as violin plots (median with interquartile range). ****P < 0.0001, NS (p = 0.1604): not significant, unpaired two-tailed Student’s t-test. d Intracellular replication rates of indicated strains with or without IAA treatment, as determined by the distribution of parasitophorous vacuoles (PVs) containing 1, 2, 4, 8 or 16 parasites after 24 h of intracellular growth. Means ± SEM of n = 3 independent experiments, each with two technical replicates, two-way ANOVA with Tukey’s multiple comparisons post-tests. e The length of trails formed by extracellular parasites gliding on bovine albumin coated surface. The parasites were pretreated with or without IAA for 48 h, mechanically released from host cells and used to glide on cover slips precoated with bovine albumin. The trails formed by parasite gliding was determined by IFA using an antibody against the surface protein SAG1. Over 150 gliding trails from n = 3 independent experiments (each contained 49 to 67 trails) were analyzed for each condition and graphed as violin plots (median with interquartile range). ***P < 0.001, unpaired two-tailed Student’s t-test. f Invasion efficiencies of the AMPKγ-mAID strain pretreated with IAA for 0, 12, 24, 48 or 72 h during the intracellular growth stage. Pretreated parasites were purified and used to invade HFF cells for 20 min and then a two-color staining assay was used to distinguish invaded parasites from non-invaded ones. Invasion efficiency of non-treated parasites (0 h) was set as 100% and used to normalize that of IAA treated parasites. Means ± SD of n = 3 independent experiments, ****P < 0.0001, unpaired two-tailed Student’s t-test, each was compared with the 0 h group. Source data are provided as a Source data file.