The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Teoh, Kim-Tat; Siu, Yu-Lam; Chan, Wing-Lim; Schlüter, Marc A.; Liu, Chia-Jen; Peiris, J. S. Malik; Bruzzone, Roberto; Margolis, Benjamin; Nal, Béatrice

MDCKII cell lines at 80–90% confluence were detached with trypsin-EDTA, resuspended in DMEM, and spun down at low speed (1200 rpm for 5 min at 4°C). The cell pellet was resuspended in DMEM containing with 200 μg/ml Hygromycin B, 600 μg/ml G418, and 4% GelTrex, and special care was paid to obtain a predominantly single cell suspension. Cyst formation assays were performed in 8-well Lab-Tek chambered slides (Nunc. Rochester, NY), which were coated with 50 μl of 100% GelTrex, a basement membrane matrix with reduced growth factor content (Invitrogen). Four thousand cells were seeded into each chamber and incubated at 37°C (95% O2/5% CO2) for 5 d to allow individual MDCKII cells to grow into cysts. Cell culture medium was changed every 2–3 d. MDCKII cysts were fixed with 4% paraformaldehyde (PFA) and immunostained as described below.