Figure S4.
Genetic manipulations of TgNup503, attempts for overexpression and generation, and analysis of inducible knock-down. (A) A scheme of the genetic manipulation designed to generate a TgNup503 overexpression line, where, using CRISPR/CAS9, a new promoter (the strong T. gondii tubulin promoter, TUB light blue) would be inserted displacing the native TgNup503 promoter (grey). The scheme also shows the location of the primers used to test for integration. (B) One example of the four genetic manipulation attempts shows a lack of promoter integration (primers 1 + 2), while primers 3 + 4 confirm successful gDNA extraction as a positive control. (C) Scheme showing the genetic manipulation to generate the inducible depletion where, using CRISPR/CAS9, the native promoter is displaced and the gene of interest (TgNup503) is now under the control of the repressible promoter (T7S4) and contains an HA tag. Gene expression can be shut down by the addition of anhydrous tetracycline (ATc). The position of the primers used in D are indicated. (D) PCR validation of the promoter integration to generate inducible TgNup503 (i-HA-TgNup503). (E) qRT-PCR showing the downregulation of TgNup503 expression 24 h post ATc addition (this experiment was performed with the parasite line resulting from the manipulation in D iHA-TgNup503 TgNup302-myc). Error bars display mean with SEM, data were analyzed with one sample T test (three biological replicates). (F) Western blot showing TgNup503 loss 24 h post ATc addition, TgCDPK1 was used as a loading control. Staining was done with secondary fluorescent antibodies followed by signal quantification. Error bars show mean with SEM, data were analyzed with two-sided unpaired T test (two biological replicates) (P = 0.0811). (G) SR image showing loss of TgNup503 24 h post ATc addition. (H) Plaque assays showing lack of growth of iTgNup503 upon TgNup503 depletion via ATc treatment. As a control, the growth of the parental line was unaffected by the presence of ATc. (I) Division assay showing growth of parental and iHA-TgNup503 TgNup302-myc line without ATc and at 16 and 24 h post addition of ATc addition. Three biological replicates, total of 300 vacuoles counted. Error bars display mean with SEM, data were analyzed with unpaired Wilcoxon test. (J) TgENO2 nuclear import assay in iHA-TgNup503 TgNup302-myc line and parental line in untreated cells and 16 and 24 h post addition of ATc. Scale bar is 2 μm. (K) PCR validation of the promoter integration to generate inducible TgNup134 (with the same strategy as shown in the scheme in C). (L) Western blot showing TgNup134 loss 24 h post ATc addition. TgCDPK1 was used as a loading control. (M) Scheme showing the genetic manipulation to generate the inducible depletion where, using CRISPR/CAS9, the native promoter is displaced, and the gene of interest (TgTom40) is now under the control of the repressible promoter (T7S4). Gene expression can be shut down by addition of anhydrous tetracycline (ATc). The position of the primers used in B are indicated. (N) PCR validation of the promoter integration to generate inducible TgTom40 (iTgTom40). (O) Western blot showing TgTom40 loss at 48 h post ATc addition, TgCDPK1 was used as a loading control. (P) Immunofluorescence images showing loss of TgTom40 at 48 h post ATc addition. (Q) Western blot showing that the mitochondrial marker TgMys remains at the same level at the 48 h time point when TgTom40 is almost fully lost. TgCDPK1 was used as a loading control. (R) Plaque assays showing lack growth of iTgTgTom40 upon TgTom40 depletion via ATc treatment. Source data are available for this figure: SourceData FS4.

Genetic manipulations of TgNup503, attempts for overexpression and generation, and analysis of inducible knock-down. (A) A scheme of the genetic manipulation designed to generate a TgNup503 overexpression line, where, using CRISPR/CAS9, a new promoter (the strong T. gondii tubulin promoter, TUB light blue) would be inserted displacing the native TgNup503 promoter (grey). The scheme also shows the location of the primers used to test for integration. (B) One example of the four genetic manipulation attempts shows a lack of promoter integration (primers 1 + 2), while primers 3 + 4 confirm successful gDNA extraction as a positive control. (C) Scheme showing the genetic manipulation to generate the inducible depletion where, using CRISPR/CAS9, the native promoter is displaced and the gene of interest (TgNup503) is now under the control of the repressible promoter (T7S4) and contains an HA tag. Gene expression can be shut down by the addition of anhydrous tetracycline (ATc). The position of the primers used in D are indicated. (D) PCR validation of the promoter integration to generate inducible TgNup503 (i-HA-TgNup503). (E) qRT-PCR showing the downregulation of TgNup503 expression 24 h post ATc addition (this experiment was performed with the parasite line resulting from the manipulation in D iHA-TgNup503 TgNup302-myc). Error bars display mean with SEM, data were analyzed with one sample T test (three biological replicates). (F) Western blot showing TgNup503 loss 24 h post ATc addition, TgCDPK1 was used as a loading control. Staining was done with secondary fluorescent antibodies followed by signal quantification. Error bars show mean with SEM, data were analyzed with two-sided unpaired T test (two biological replicates) (P = 0.0811). (G) SR image showing loss of TgNup503 24 h post ATc addition. (H) Plaque assays showing lack of growth of iTgNup503 upon TgNup503 depletion via ATc treatment. As a control, the growth of the parental line was unaffected by the presence of ATc. (I) Division assay showing growth of parental and iHA-TgNup503 TgNup302-myc line without ATc and at 16 and 24 h post addition of ATc addition. Three biological replicates, total of 300 vacuoles counted. Error bars display mean with SEM, data were analyzed with unpaired Wilcoxon test. (J) TgENO2 nuclear import assay in iHA-TgNup503 TgNup302-myc line and parental line in untreated cells and 16 and 24 h post addition of ATc. Scale bar is 2 μm. (K) PCR validation of the promoter integration to generate inducible TgNup134 (with the same strategy as shown in the scheme in C). (L) Western blot showing TgNup134 loss 24 h post ATc addition. TgCDPK1 was used as a loading control. (M) Scheme showing the genetic manipulation to generate the inducible depletion where, using CRISPR/CAS9, the native promoter is displaced, and the gene of interest (TgTom40) is now under the control of the repressible promoter (T7S4). Gene expression can be shut down by addition of anhydrous tetracycline (ATc). The position of the primers used in B are indicated. (N) PCR validation of the promoter integration to generate inducible TgTom40 (iTgTom40). (O) Western blot showing TgTom40 loss at 48 h post ATc addition, TgCDPK1 was used as a loading control. (P) Immunofluorescence images showing loss of TgTom40 at 48 h post ATc addition. (Q) Western blot showing that the mitochondrial marker TgMys remains at the same level at the 48 h time point when TgTom40 is almost fully lost. TgCDPK1 was used as a loading control. (R) Plaque assays showing lack growth of iTgTgTom40 upon TgTom40 depletion via ATc treatment. Source data are available for this figure: SourceData FS4.

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