After exposure to 10 µM D3/EMEM (treatment group) and the same concentration of ethanol/EMEM (vehicle control group), the cells were incubated in ice-cold RIPA Lysis Buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulphate, 1% sodium deoxycholate; 10% EDTA solution, 10% protease and phosphatase inhibitor cocktail; Abcam, Cambridge, MA, USA) for 30 minutes, then centrifuged at 13,200 rpm to obtain the supernatants, which were frozen at −80°C. Protein concentrations were determined with the Pierce BCA protein assay kit (Thermo Fisher Scientific). Protein samples were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes, which were then probed overnight at 4°C with primary antibodies against human α smooth muscle actin (α-SMA; ab5694, 1 µg/mL; Abcam), vimentin (EPR3776; ab92547, 1:5,000; Abcam), and GADPH (sc-47724, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubating with horseradish peroxidase–conjugated secondary antibodies (A16078, 1:20,000, Invitrogen; A6154, 1:20,000, Sigma-Aldrich Corp.) for two hours at room temperature. Labeled proteins were detected by enhanced chemiluminescence (no. 34579, Thermo Fisher Scientific).