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Fig. S1. Analysis of IRG pixel intensity on T. gondii PVs.
Details of this analysis are given in Experimental procedures. To measure the intensity of IRG signal on T. gondii PVs two lines were drawn approximately at right angles across each vacuole (A). Each line thus crossed the ‘margin’ of the vacuole twice, giving a total of four values for each vacuole using the Image J and Axiovision 4.7 software. The pixel intensity of each vacuole was defined as the mean of these four values after background subtraction (B). To investigate how much variation in signal intensity measurements is caused by inhomogeneity of the IRG signal around the rim of the PV, 20 profiles were created by drawing 20 parallel lines through each of three more or less brightly Irgb6- positive vacuoles (stained by serum A20). The mean values of pixel intensity were calculated from each profile and presented as single dots and the arithmetic means are given as horizontal lines (C). Variation of Irgb6 intensity around the rim of single PVs clearly does not account for the total heterogeneity of IRG protein signal shown on and elsewhere as the different signal intensities on the three vacuoles can be clearly distinguished. (D) Selected frames of two time-lapse videos of Irgb6- FLAG-EGFP loading on ME49 T. gondii PV. MEFs were transfected with the expression plasmid pEGFP-N3-Irgb6-FLAG and simultaneously induced with IFNγ. After 24 h, the cells were infected with T. gondii ME49 strain in microscope slide chambers as described in Zhao et al. (2009a) and monitored continuously in order to document the entry of individual parasites and the subsequent accumulation of Irgb6-FLAG-EGFP on the PV. Arrowheads indicate the location of the analysed T. gondii PVs.
Fig. S2. Inhibition of major signalling pathways and microtubule polymerization.
A. The blockade of PI3 kinase and G protein-coupled receptors was demonstrated by Western blot for phospho-Akt (pAkt). MEFs were treated with wortmannin (W), LY294002 (LY) or pertussis toxin (PT) for 6 h as described in Experimental procedures. Inhibited cells were stimulated by EGF for 10 min to induce Akt phosphorylation. Calnexin and total Akt served as a loading control. B. To inhibit caspases MEFs were treated with z-VAD-fmk for 2 h followed by stimulation with TNFα for 6 h in the presence of cycloheximide (Chx), as described in Experimental procedures. The amount of PARP1 processing by caspases was analysed by Western blot. Calnexin served as a loading control.
C. Inhibition of microtubule polymerization was monitored microscopically by immunostaining with an anti-α-tubulin mouse monoclonal antibody. MEFs were induced with IFNγ for 24 h and treated with nocodazole, or DMSO as control, for 1 h, as described in Experimental procedures. Treated cells were infected with T. gondii ME49 strain and loading of parasite PVs was monitored 2 h after infection by immunostaining for Irgb6 (serum A20).
Fig. S3. Irga6 aggregates do not colocalize with lysosomal marker LAMP1 in Atg5-/- fibroblasts. Cells were prepared as described in and immunostained for Irga6 (mAb 10D7) and LAMP1 (mAb 1D4B). No obvious colocalization of Irga6 with LAMP1 in wt and Atg5-/- fibroblasts was observed. Arrows indicate intracellular parasites identified by phase contrast (PhC), and arrowheads indicate Irga6 aggregates.
Fig. S4. Frequencies of IRG protein-positive vacuoles detected by specific antibody reagents at different dilutions: saturation of frequency estimates. IFNγ-induced C57BL/6 MEFs were infected with T. gondii ME49 for 2 h. IRG proteins were detected by serial twofold dilution of antibody reagents around the dilutions typically used for experiments followed by an appropriate secondary reagent to estimate loaded vacuole frequencies. The experimental dilutions for each reagent were as follows: for Irgb6: mAb B34 at 1/1000 (3.4 mg ml-1 stock), serum A20 at 1/200; for Irgb10: serum anti-Irgb10 at 1/2000; for Irga6: mAb 10E7 at 1/1000 (3.7 mg ml-1 stock), mAb 10D7 at 1/2000 (4.3 mg ml-1 stock), serum 165/3 at 1/8000; for Irgd: serum 081/1 at 1/4000. The most concentrated dilution is designated as 1 (mAb B34 at 1:250, serum A20 at 1:50, serum anti-Irgb10 at 1:500, mAb 10E7 at 1:100, mAb 10D7 at 1:500, serum 165 at 1:2000, serum 081/1 at 1:4000). IRG-positive PVs were counted blind from 100–200 intracellular parasites. Frequency estimates for loaded vacuoles were independent of antibody or antiserum dilution in the range tested.
Fig. S5. Irga6-ctag1-EGFP loads before Irgd-ctag1-Cherry onto T. gondii ME49 strain PV. C57BL/6 MEFs were induced with IFNγ and transfected simultaneously with constructs expressing Irga6- ctag1-EGFP and Irgd-ctag1-Cherry. After 24 h, cells were infected with T. gondii ME49 strain in microscope slide chambers and observed by live cell imaging for the accumulation of IRG proteins. Successive 3 min frames from one vacuole (arrow) show Irga6 visibly loading several frames before Irgd.
Fig. S6. Irga6 and Irgb6 pixel intensity profiles of RH-YFP PVs in IFNγ-induced cells. IFNg-induced MEFs were infected with the T. gondii strain RH-YFP for 2 h. The pixel intensities of the Irga6 (mAb 10D7, red) and serum Irgb6 (A20, blue) signals at the RH-YFP (green) PVM across transects of the vacuole are displayed as a function of distance (μm). Five representative PVs of the three categories Irga6 and Irgb6 double-positive (A), Irga6 single-positive (B) and double-negative (C) are given (see also ). The baseline of the A20 serum staining is high due to diffuse background staining of the blue second stage reagent.
Fig. S7. IFNγ-restrained proliferation of T. gondii is not affected by the virulence-associated ROP proteins, ROP5 and ROP16. The growth of RH-Δrop16, S22-LC37 (carrying the cosmid expressing 4 ROP5 and 2 other genes) and control RH and S22 T. gondii strains was measured by 3H-uracil incorporation as described in Experimental procedures. The data are presented in radioactive counts per 3 min. Black, light grey and dark grey bars indicate proliferation of the parasite in MEFs untreated, stimulated with 10 U ml-1 and 100 U ml-1 of IFNγ respectively.
Table S1. Summary of experiments analysing the correlated loading of selected pairs of IRG proteins on to the ME49 PV.
Video S1. Time-lapse video of Irga6-ctag1-EGFP association with the T. gondii PV. IFNγ-treated MEFs were transfected with the construct expressing Irga6-ctag1-EGFP and infected with T. gondii ME49. Time-lapse video was started immediately after infection and the images were collected every 30 s (see also ). The video is presented at 6 frames per second.
Video S2. Time-lapse video of Irga6-ctag1-EGFP association with the T. gondii PV. IFNγ-treated MEFs were transfected with the construct expressing Irga6-ctag1-EGFP and infected with T. gondii ME49. Time-lapse video was started 1 h after infection and the images were collected every 3 min (see also ). The video is presented at two frames per second. Out of focus frames were excluded from the movie.
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