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

FIG 1 .

FIG 1 

Recruitment of autophagy adaptors to intracellular T. gondii. (A to D) Quantification of recruitment of autophagy adaptors to type 1 (GT-1), 2 (PTG), or 3 (VEG) T. gondii in HeLa cells activated for 24 h with IFN-γ prior to infection and analyzed at 6 h postinfection. Data points represent the mean red fluorescence of the respective host markers in a ROI overlapping the T. gondii vacuole. Arrowheads indicate the mean red fluorescence value of 90% of visually determined positive vacuoles. Each value is the mean ± SEM of three experiments (***, P ≤ 0.001; *, P ≤ 0.05; Kruskal-Wallis test). (E) Immunofluorescence localization of ubiquitin, p62, NDP52, or LC3 to the PVM in HeLa cells activated with 5 ng/ml IFN-γ at 6 h postinfection with type 3 (VEG) parasites. Ubiquitin was localized with mouse MAb FK2, followed by anti-mouse IgG conjugated to Alexa Fluor 594 (red). p62 was localized with a guinea pig polyclonal antibody, followed by anti-guinea pig IgG conjugated to Alexa Fluor 594 (red). NDP52 was localized with a rabbit polyclonal antibody, followed by anti-rabbit IgG conjugated to Alexa Fluor 594 (red). LC3 was localized with a rabbit polyclonal antibody, followed by Alexa Fluor 594 (red). T. gondii PVM was localized with either a rabbit polyclonal antibody to GRA7, followed anti-rabbit IgG conjugated to Alexa Fluor 488 (green), or mouse MAb tg17-113 to GRA5, followed by anti-mouse IgG conjugated to Alexa Fluor 488 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 5 µm. (F) Visual assessment of the percentage of host marker-positive T. gondii PVs from images used to collect the data shown in panels A to D. Each value is the mean ± SEM of three experiments. (G) Quantification of recruitment of autophagy proteins to type 3 (VEG) parasites in naive or IFN-γ-activated HeLa cells at 6 h postinfection by fluorescence microscopy. There were two experiments and a total of six coverslips. Each value is the mean ± SD. (H) Immunofluorescence images of HeLa cells infected with type 3 (VEG) parasites at 6 h postinfection. HeLa cells were activated with 5 ng/ml IFN-γ. Ubiquitin was localized with mouse MAb FK2, p62 was localized with a guinea pig polyclonal antibody, NDP52 was localized with a rabbit polyclonal antibody, and LC3 was localized with a rabbit polyclonal antibody, followed by secondary antibodies conjugated to Alexa Fluor 488 (green) or 594 (red), as indicated. T. gondii PVM was localized with either a rabbit polyclonal antibody to GRA7 or mouse MAb tg17-113 to GRA5, with the opposite antibody species to the host marker, followed by anti-mouse IgG conjugated to Alexa Fluor 488 (green) or 549 (red). Nuclei were stained with DAPI. Scale bar, 5 µm. See also Fig. S1 in the supplemental material.