Immunofluorescence microscopy
Caco-2 cells seeded on glass coverslips were infected with SARS-CoV-2 Isolate Muc-IMB-1/2020, second passage on Vero E6 cells (2x106 PFU/mL) at an MOI of 0.1 or 0.01. At 24 hours post-infection cells were washed with PBS and fixed in 4% paraformaldehyde in PBS for 20 minutes at RT, followed by permeabilization with 0.3% Triton X-100 in PBS for 10 minutes at RT and blocking in 5% fetal calf serum in PBS for 1 hour at RT. Incubation with primary antibodies against CK2α (Abcam, ab70774, 1:500), SARS-CoV membrane (M) protein (Rockland, #100-401-A55, 1:500) and SARS-CoV nucleocapsid (N) protein (Rockland, #200-401-A50, 1:1000) was performed for 1 hour at RT. After washing with PBS, cells were incubated with AF568-labeled goat-anti-rabbit (Invitrogen, #A11011) and AF647-labeled goat-anti-mouse (Invitrogen, #A21235) secondary antibodies (1:200) as well as AF488-labeled Phalloidin (Hypermol, #8813-01, 1:250) for 2 hours at RT. Fluorescence images were generated using a LSM800 confocal laser-scanning microscope (Zeiss) equipped with a 63X, 1.4 NA oil objective and Airyscan detector and the Zen blue software (Zeiss) and processed with Zen blue software and ImageJ/Fiji. For 3D-reconstruction, cells were fixed and stained as indicated and imaged as z stack with 0.15 μm sections. Z stack was processed using Imaris software 64x 9.5.1 and displayed as MIP. To quantify colocalization between casein kinase II and N protein colocalization events in filopodia of SARS-CoV-2 infected Caco-2 cells were counted. 42 ± 19 % of the N protein particles detected in filopodia colocalize with CK2. Length of filopodia was measured using Metamorph (Version 7.8). Distance was measured starting at cortical actin to the tip of Filopodia.