Knockout of TgTEP does not affect the localization of micronemes, rhoptries, dense granules, IMC, or apicoplast but causes mitochondrial fragmentation. (A–E) IFAs using antibodies against (A) micronemes (Mic8), (B) rhoptries (Rop2,4), (C) dense granules (Gra1), (D) IMC (IMC1), and (E) apicoplast (G2-Trx) (all in yellow) showed that knockout of TgTEP (tagged with mCherry, shown in magenta) has no effect on their localization. Immunofluorescence assays were done in triplicate, with a minimum number of 100 vacuoles per replicate observed. The nuclei were labelled with Hoechst. (F) Knockout of TgTEP (in magenta) results in the fragmentation of the mitochondria (marked using the anti-TOM40 antibodies in yellow). All scale bars are 5 μm. (G) Quantification of mitochondrial fragmentation at different time points after induction of TgTEP knockout. Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was done, with P values being represented as follows: ns ≥ 0.05; * = 0.01–0.05; **** ≤0.0001. (H) The percentage of parasitophorous vacuoles showing altered CPL localization was significantly higher in knockout mutants after 48 h post-induction (hpi) with 50 nM rapamycin. The assay was done three times, with a minimum of 100 vacuoles quantified per condition per replicate. The data are plotted as mean ± SD. Besides, CPL signal intensity quantifications confirmed an accumulation of CPL, this being significantly higher in knockout parasites after 48 h post-induction (hpi) with rapamycin. The assay was done three times, with the intensities of a minimum of 15 vacuoles quantified per condition per replicate. The data are plotted as mean ± SD, with the dots representing the value for each data point. For both assays, one-way ANOVA with Tukey’s multiple comparison test was done. The P values are represented as follows: ns ≥ 0.05; ** = 0.001–0.01; **** ≤0.0001. (I) CPL signal, which typically appears more confined to a few structures in extracellular WT parasites, also appeared to accumulate in extracellular TgTEP-knockout parasites. Scale bars are 3 μm.
Figure S2.

Knockout of TgTEP does not affect the localization of micronemes, rhoptries, dense granules, IMC, or apicoplast but causes mitochondrial fragmentation. (A–E) IFAs using antibodies against (A) micronemes (Mic8), (B) rhoptries (Rop2,4), (C) dense granules (Gra1), (D) IMC (IMC1), and (E) apicoplast (G2-Trx) (all in yellow) showed that knockout of TgTEP (tagged with mCherry, shown in magenta) has no effect on their localization. Immunofluorescence assays were done in triplicate, with a minimum number of 100 vacuoles per replicate observed. The nuclei were labelled with Hoechst. (F) Knockout of TgTEP (in magenta) results in the fragmentation of the mitochondria (marked using the anti-TOM40 antibodies in yellow). All scale bars are 5 μm. (G) Quantification of mitochondrial fragmentation at different time points after induction of TgTEP knockout. Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was done, with P values being represented as follows: ns ≥ 0.05; * = 0.01–0.05; **** ≤0.0001. (H) The percentage of parasitophorous vacuoles showing altered CPL localization was significantly higher in knockout mutants after 48 h post-induction (hpi) with 50 nM rapamycin. The assay was done three times, with a minimum of 100 vacuoles quantified per condition per replicate. The data are plotted as mean ± SD. Besides, CPL signal intensity quantifications confirmed an accumulation of CPL, this being significantly higher in knockout parasites after 48 h post-induction (hpi) with rapamycin. The assay was done three times, with the intensities of a minimum of 15 vacuoles quantified per condition per replicate. The data are plotted as mean ± SD, with the dots representing the value for each data point. For both assays, one-way ANOVA with Tukey’s multiple comparison test was done. The P values are represented as follows: ns ≥ 0.05; ** = 0.001–0.01; **** ≤0.0001. (I) CPL signal, which typically appears more confined to a few structures in extracellular WT parasites, also appeared to accumulate in extracellular TgTEP-knockout parasites. Scale bars are 3 μm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal