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. 2015 Sep 8;6(5):e01157-15. doi: 10.1128/mBio.01157-15

FIG 6 .

FIG 6 

Replication of T. gondii in autophagy-deficient HeLa cells. (A) Replication of type 3 (VEG) T. gondii in vacuoles positive for ubiquitin in IFN-γ-activated wild-type (WT), ATG16L1 KO (clone E6), or ATG16L1-complemented (WT/Comp or E6/comp) host cell strains at 24 h postinfection. Each value is the mean ± SEM of three experiments (***, P < 0.001; two-way ANOVA). (B) Representative fluorescence images of T. gondii vacuoles in ATG16L1 KO or complemented strains. Ubiquitin was localized with mouse MAb FK2, followed by goat anti-mouse IgG conjugated to Alexa Fluor 594 (red). Parasites were localized with a rabbit polyclonal antibody to tachyzoites (αTg), followed by goat anti-rabbit IgG conjugated to Alexa Fluor 488 (green). Scale bars, 5 µm. (C) Replication of type 3 (VEG) T. gondii positive for ubiquitin in IFN-γ-activated ATG7 KO HeLa cells at 24 h postinfection. Each value is the mean ± SEM of three experiments (***, P < 0.001; two-way ANOVA). (D) Representative images of T. gondii vacuoles in wild-type (WT) HeLa cells or ATG7 KO clones (1A6 or 1A11). Ubiquitin was localized with mouse MAb FK2, followed by goat anti-mouse IgG conjugated to Alexa Fluor 594 (red). Parasites were localized with a rabbit polyclonal antibody to tachyzoites (αTg), followed by goat anti-rabbit IgG conjugated to Alexa Fluor 488 (green). Scale bars, 5 µm. (E) Quantification of recruitment of ubiquitin to type 3 (VEG) parasites in IFN-γ-activated (5 ng/ml) U2OS cells treated with tetracycline-inducible shRNA in the presence or absence of tetracycline at 6 h postinfection. Each value is the mean ± SEM of three experiments (n.s., not significant; two-way ANOVA). (F) Expression of Beclin 1 and ATG14 detected by Western blotting of lysates from parental U2OS cells or ± tetracycline-induced shRNA knockdown of Beclin 1 and ATG14. Beclin 1 and ATG14 were detected with a rabbit MAb (Beclin 1) or a rabbit polyclonal antibody (ATG14), followed by LiCor IRDye 800CW goat anti-rabbit IgG (green). Actin was used as a loading control and detected with mouse MAb C4, followed by LiCor IRDye 680CW goat anti-mouse IgG (red). Arrowheads indicate Beclin 1 or ATG14 protein. The values to the left are molecular sizes in kilodaltons. (G) Quantification of recruitment of ubiquitin to type 3 (VEG) parasites in naive or IFN-γ-activated HeLa cells treated with 25 µM Tat-Beclin 1, Tat-scrambled peptide, or Opti-MEM at 6 h postinfection. Each value is the mean ± SD of two experiments with three internal replicates and a total of six coverslips. n.s., not significant; Kruskal-Wallis test. (H) Representative fluorescence images of HeLa cells expressing LC3-GFP treated with 25 µM Tat-Beclin 1 or Tat-scrambled peptide for 3 h. GFP-LC3 was localized with mouse MAb 3E6 against GFP, followed by goat anti-mouse IgG conjugated to Alexa Fluor 488. Scale bar, 20 nm. See also Fig. S6 in the supplemental material.