FIG 4.

Loss of TgMAPK2 leads to a defect in daughter cell budding. TgMAPK2^AID parasites stably expressing mTFP1–α-tubulin (magenta) and TgIMC1-mVenus (green) were stained with Hoechst 33342 (blue) to distinguish the three indicated categories in the cell cycle. Bars = 2 μm. (B) Experimental flow of the addition of IAA in increasing 2-h increments. (C) Quantification of parasites in each category with increasing time of growth in IAA. Shown are means ± SD from 3 biological replicates; 200 to 300 parasites were counted under each condition. (D) Quantification of parasites with daughter cell budding rings (ISP1 in daughter buds) grown in the presence or absence of IAA for 8 h. Means ± SD from 3 biological replicates are shown; 200 to 300 parasites were counted under each condition. P values are from two-tailed Student’s t test. (E) Western blot demonstrating that MAPK2^AID protein levels are restored 2 to 4 h after IAA washout. (F) Phase-contrast micrograph comparing the growth of TgMAPK2^AID parasites over 18 h in IAA or after 2 h in IAA with an additional 16 h after washout. Arrowheads indicate individual vacuoles. Bars = 10 μm. (G) Quantification of the percentage of vacuoles that appeared to be replicating normally (≥4 parasites/vacuole) from 3 independent replicates. P values are from one-way analysis of variance (ANOVA) with Dunnett’s test (***, P < 0.0001). w-o, without.