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. 2022 Dec 8;13:7560. doi: 10.1038/s41467-022-35267-5

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a The indicated parasite strains were allowed to infect HFF cells for 4 h followed by incubation in an alkaline culture medium without CO₂ for 4 days for induction of bradyzoites. Parasites were stained with anti-IMC1 antibody (red) and bradyzoite cyst wall was detected by FITC-Dolichos biflorus lectin (DBL) (green). PruΔBfd1 was used as a negative control for stage conversion. Scale bar, 10 µm. b Quantification of differentiation in wild-type, knockout, and complemented strains following 4 days of exposure to alkaline stress. The mean ± SD was plotted for six biological replicates, with percentage of DBL positive vacuoles calculated based on at least 100 vacuoles per replicate. Statistical significance tested by Student’s one-tailed t test and the indicated strains were compared with Pru strain ****P value < 0.0001, n.s. = not significant. c Western blotting analysis of the tachyzoites lysates (incubated for 2–3 days under normal culture conditions) and bradyzoites (incubated for 4 days under high alkaline culture conditions) by anti-BAG1 antibody. d The indicated parasites were added to HFF cells for 4 h to allow the parasites to invade host cells, followed by incubation in an alkaline culture medium without CO₂ for 4 days for induction of bradyzoites. Parasites were detected by anti-GAP45 antibody (green) and bradyzoite cysts were stained with succinylated wheat germ agglutinin (s-WGA) (red) and the bradyzoites specific anti-BAG1 antibody (magenta). Scale bar, 10 µm. e Transmission electron micrograph of parasites incubated for 4 days under high alkaline culture conditions. Arrowheads point to cyst wall (CW) or parasitophorous vacuole membrane (PVM) in the insert. f Quantification of spontaneously differentiated bradyzoites of Pru and PruΔpp2a-c strains following 48 h growth in differentiated myotubes. The mean ± SD was plotted for three biological replicates, with the percentage of DBL positive vacuoles calculated based on examining at least 100 vacuoles per sample. (Student’s one-tailed t test, *P value = 0.0159). Source data are provided as a Source data file.