(A) Astrocytes were induced with the indicated concentrations of IFN-γ and infected with T. gondii 24 h later for 68 h. The growth of intracellular parasites was monitored by uracil incorporation assay. (Inset) Lysates of astrocytes induced with the indicated concentrations of IFN-γ for 24 h were probed for IIGP1 protein by Western blotting.
(B) Untreated or IFN-γ induced astrocytes were infected with T. gondii. After 2 h, extracellular parasites were washed away and cells were either fixed or incubated further for a total of 8 h or 24 h. Shown are the mean values of three independent counts representing a total number of 650–997 cells per time point.

(A–D) IFN-γ-induced astrocytes were infected with T. gondii for 2 h, fixed, and stained for IGTP (A), GTPI (B), TGTP1 (C), or IRG-47 (D).
(E) IFN-γ-induced astrocytes were infected with T. gondii for 2 h, fixed, and stained for IIGP1 (red) and T. gondii (green).
(F) IFN-γ-induced cells were infected with T. gondii, fixed 24 h later, and stained for IIGP1. White arrowheads point to PVs containing replicating parasites.
(G and H) IFN-γ-induced cells were infected with heat-killed (G) or live (H) parasites, fixed 2 h later, and stained for IIGP1 (green) and LAMP1 (red). White arrowheads in (G) point to parasites residing in a LAMP1-positive but IIGP1-negative compartment. (H) Shows single sections of a 3D deconvoluted Z-series. Nuclei of host cells and parasites were stained with DAPI.

Astrocytes were induced with IFN-γ or left untreated and infected with T. gondii 24 h later for 2 h. Cells were fixed and stained for the indicated proteins. (A) Shows a cell that was stained for IIGP1 (red) and calnexin (green). The vacuolar calnexin signal is markedly less concentrated at the PV than IIGP1. (A') shows the same cell as in (A) but with an electronically enhanced IIGP1 signal to reveal its non-vacuolar ER localization. Note the dramatic difference in the ratio of the ER versus PV signal between IIGP1 and calnexin.
(B and C) Shows astrocytes stained for IIGP1- and the ER-localized PDI. No PDI accumulation at the PV was detected.
(D) Astrocytes were treated as above but stained for IIGP1 (red) and ERP60 (green). Nuclei were stained with DAPI.

(A) IFN-γ-induced astrocytes were infected with T. gondii for 6 h, fixed, and subjected to ultra-thin cryosectioning. Sections were labeled for IIGP1 using the 165 antiserum and 10 nm gold particles coupled to protein A. The right side is an enlarged view of the boxed region showing that the IIGP1 label was found in close proximity to the PVM (black arrowhead: PVM; white arrowhead: T. gondii plasma membrane; open arrowhead: T. gondii inner membrane complex; bars 200 nm and 100 nm [inset]).
(B) IFN-γ-induced astrocytes were fixed at the indicated times post-infection (MOI of 10) and 110–160 IIGP1-positive vacuoles were counted per time point. Shown is the percentage of smooth (white), rough (hatched), and disrupted vacuoles (black).
(C) IFN-γ-induced astrocytes were infected with T. gondii, fixed 2 h later, and stained for IIGP1 with the 10D7 monoclonal antibody (left) or the 165 antiserum (right). The images show maximum projections of 3D deconvoluted Z-series.

(A and B) IFN-γ-induced astrocytes were infected with T. gondii for 2 h (A) or 6 h (B) and stained for IIGP1 (green) and GRA7 (red) (filled arrowheads: IIGP1-negative PVs, open arrowheads: IIGP1-positive PVs). Nuclei were stained with DAPI. (C) Uninduced (left) and IFN-γ-induced (right) astrocytes were infected with T. gondii and stained for GRA7 at 4 h post-infection. Exposure conditions for the two images were the same.

Astrocytes were induced with IFN-γ, infected with T. gondii and fixed 6 h later. (A–C) Ultra- thin cryosections were labeled for IIGP1 using the 165 antiserum and 10 nm gold coupled to protein A. The insets in (A) show enlarged views of the boxed regions. The black arrows in the bottom left inset point to IIGP1-labeled vesicular profiles with an apparent electron-dense coat. (D and E) Astrocytes were labeled with the anti-GRA7 mAb and 10 nm gold coupled to protein A. (open white arrowhead: T. gondii inner membrane complex [IMC]; filled white arrowhead: T. gondii plasma membrane; black arrowhead: PVM). Bars: 250 nm.

(A) Astrocytes were transfected with IIGP1ctag1 and simultaneously induced with IFN-γ. After 24 h, cells were infected with T. gondii, fixed 2 h later, and stained for ctag1 (green) and IIGP1 (red).
(B) Cells were treated as in (A) but transfected with IIGP1K82Actag1. White arrowheads point to T. gondii-containing vacuoles.
(C) Astrocytes were treated as in (A), fixed at the indicated time points, and the number of vacuoles displaying a smooth, rough, or disrupted morphology was counted. Shown are the mean values of two independent experiments. (White bars: smooth vacuoles; hatched bars: rough vacuoles, black bars: disrupted vacuoles)
(D) Astrocytes were stimulated with IFN-γ and transfected with IIGP1ctag1 (black bars) or IIGP1K82Actag1 (white bars). Cells were infected with T. gondii 24 h later and fixed at the shown time points. Three independent experiments (A–C) are shown. 217–570 cells per time point and condition were counted.
(E) Astrocytes isolated from neonatal IIGP1^−/− mice or wild-type littermates were induced with the indicated concentrations of IFN-γ and infected with T. gondii 24 h later for a total of 48 h. The growth of intracellular parasites was monitored by uracil incorporation assay (p values: ** 0.0015, *** 0.0001 (unpaired Student t-test)).

Astrocytes were transfected with pEGFP-C3-LC3 and induced with IFN-γ. Cells were infected with T. gondi 24 h later and fixed after 2 h (A) or 6 h (B, C, and D). In cells containing only smooth IIGP1 vacuoles GFP-LC3 remained diffusely distributed throughout the cytoplasm (A). In cells containing disrupted IIGP1 PVs GFP-LC3 localizes to vesicular and filamentous structures that are in close proximity to, but do not engulf the IIGP1-positive PVs (B and C). The arrowheads point to IIGP1-positive PVs. The images shown in (A) were processed by 2D deconvolution. (D) Shows maximum projections of 3D deconvoluted Z-series.