Planapo 40 0.95 objective

Cells were seeded onto glass slides by CYTOSPIN 4 (Thermo Fisher Scientific) and stained with May-Grunwald and Giemsa staining solution (Merck KGaA, Germany) in accordance with the manufacturer’s instructions. The slides were examined using a BIOREVO BZ-9000 (KEYENCE, Japan). A PlanApo 40×/0.95 objective (Nikon, Japan) and the BZ-II Viewer software program (KEYENCE) were used for the image acquisition.

HEK293 cells were transfected for 24 h with mTagRFP‐Membrane‐1 that was a gift from Michael Davidson (Addgene plasmid # 57992). 3 h before imaging, cells were checked for an even membrane fluorescence and reseeded to obtain single cells attaching to the surface. Z‐stacks of 12‐bit images were taken at a Nikon A1R confocal microscope (Japan) using a Nikon Plan Apo 40×/0.95 objective, with image size of 1024 × 1024 pixels, pixel dwell time of 1.1 µs, pixel size = 0,31 µm per px, Z‐step = 0.374 µm and four times averaging. The Amira software 6.5.0 (Thermo Fisher Scientific) was used for 3D convolution, mesh generation, and surface area calculation.

Host cells were seeded and pre-stimulated as above in 96-well imaging plates (Ibidi). Cells were infected with 80,000 parasites/well and centrifuged at 300 g for 5 min to synchronise infection. At 3 h post-infection, cells were washed 3× in DPBS to remove extracellular parasites, then fixed in 4% PFA for 15 min and blocked with 3% BSA in DPBS for 1 h at room temperature. Extracellular parasites were stained prior to permeabilisation using 1:1,000 rabbit anti-toxo (Abcam, ab138698) and 1:500 goat anti-rabbit-AlexaFluor647 (Life Technologies, A21244). Cells were then permeabilised with 0.2% Triton-X100 for 10 min and re-blocked with 3% BSA. Host marker recruitment was probed for 2 h at room temperature using the following antibodies: mouse anti-total ubiquitin (1:200, Merck, ST1200), rabbit anti-K63 ubiquitin (1:100, Merck, 05–1308), rabbit anti-K48 ubiquitin (1:500, Sigma, ZRB2150), rabbit anti-M1 linear ubiquitin (1:200, Sigma, ZRB2114), or rabbit anti-RNF213 (1:1,000, Sigma, Human Protein Atlas no. HPA003347), followed by donkey anti-mouse-AlexaFluor488 (Thermo, A32766) or donkey anti-rabbit-AlexFluor488 (Thermo, A32790) for 1 h at room temperature. For measurement of K63 recruitment, cells were imaged on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon Plan APO 40×/0.95 objective, with at least 100 intracellular vacuoles scored/condition.