The following reagents were used in this study: blebbistatin (Thermo Fisher Scientific) used at 25 μM, HGF (R&D Systems) used at 25 ng/ml, CK666 (Sigma) used at 200 μM, and CK869 (Sigma) used at 30 μM. Cells were treated with the indicated inhibitors for 1–2 h at 37°C. For hypo-osmotic media, regular media was diluted 1:5 with H2O, as previously reported (17 ). For hyperosmotic solutions, 300 mM sorbitol (MilliporeSigma) was applied.
MDCK I and II cells (23 (link),24 (link)) are maintained by the European Collection of Authenticated Cell Cultures (ECACC) and were purchased through MilliporeSigma. Human foreskin fibroblasts (HFFs) and parental MDCK (NBL-2) cells were purchased from the American Tissue Type Collection (ATCC). All cells were used at passages 8–25 and were maintained in phenol red-free DMEM (HyClone) containing 7.5% FBS (MilliporeSigma), 4.5 g/liter glucose, 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen), and 2mM L-glutamine (Life Technologies) at 37°C and 10% CO2.
MDCK I and II cells (23 (link),24 (link)) are maintained by the European Collection of Authenticated Cell Cultures (ECACC) and were purchased through MilliporeSigma. Human foreskin fibroblasts (HFFs) and parental MDCK (NBL-2) cells were purchased from the American Tissue Type Collection (ATCC). All cells were used at passages 8–25 and were maintained in phenol red-free DMEM (HyClone) containing 7.5% FBS (MilliporeSigma), 4.5 g/liter glucose, 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen), and 2mM L-glutamine (Life Technologies) at 37°C and 10% CO2.