96 well imaging plates

Host cells were seeded and pre-stimulated as above in 96-well imaging plates (Ibidi). Cells were infected with 400,000 parasites/well and centrifuged at 300 g for 5 min to synchronise infection. At 30 min post-infection, cells were washed 3× in DPBS to remove uninvaded parasites, and then 5 μm Compound 2 (gift from Michael Blackman) was added to treated wells. Cells were returned to the incubator for a further 2.5 h, then fixed and stained for total ubiquitin as described above.

Twenty-four hours after transfection, HUVECs were re-seeded in 96-well imaging plates (Ibidi) to reach ~80% confluence at 48 h after transfection. For EdU staining, cells were cultured in EGM2 media containing 10 μM EdU (Thermo) for 3 h prior to fixation at 37 °C and in 5% CO₂. Cells were fixed in 4% PFA for 15 min at room temperature and then permeabilised with 0.5% Triton X-100 for 15 min. To prevent antibody binding to non-specific epitopes, blocking was performed for 1 h in 5% goat serum, 1% BSA and 0.1% Triton X-100. Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C. The following primary antibodies were used: phospho-Histone H3 (Cell Signaling Technology, #9713, 1:500) and J2A (Scicons, 1:200). Alexa Fluor-conjugated secondary antibodies were used for detection (Thermo, 1:250). The Click-iT EdU Alexa Fluor 647 imaging kit was used for detection of EdU incorporation (Thermo). Imaging was performed using an LSM800 Observer (Zeiss).

Host cells were seeded to confluency in 96-well imaging plates (Ibidi). Cells were pre-stimulated for 24 h with 100 U/ml IFNγ according to host species. Cells were infected at an MOI of 0.3 for 24 h, then fixed in 4% PFA and stained with 5 μg/ml DAPI and 5 μg/ml CellMask Deep Red (Invitrogen). Plates were imaged using the Opera Phenix high-content screening system, with 25 images and 5 focal planes acquired per well. Automated analysis of infection phenotypes was performed using Harmony v5 (PerkinElmer) as described in [54 ]. Data is reported as the mean proportion of each factor (total Toxoplasma number, vacuole size or vacuole number) in IFNγ-treated wells relative to untreated wells. Differences between strains were determined by paired two-sided t test.