Sicav1

After attachment, primary hepatocytes were cultured overnight with 10 nM siRNA targeting CAV1 (siCAV1, Dharmacon, Thermo Fisher Scientific, United States) and negative control siRNA (siCon, SI03650318, Qiagen) using RNAiMAX transfection reagent (Invitrogen, Darmstadt, Germany) according to the manufacturer’s instructions. Next day, the hepatocytes were treated with 5 ng/ml recombinant TGF-β1 (Peprotech, Hamburg, Germany) and cultured additional 48 h in starvation medium. For AML12 cells, 10 nM siCon and siCAV1 was transfected to the cells with Lipofectamine RNAiMAX reagent after attachment, and incubated for 24 h. Afterward, cells were cultured in starvation medium for 12 h, and subsequently treated with 5 ng/ml recombinant TGF-β1 for 24 h.

The small interfering (si)RNA that were designed to specifically target the coding region of Cav1 (siCav1) mRNA was purchased from Dharmacon Inc (Lafayette, CO). Scrambled control siRNA (C‐siRNA), which had no sequence homology to any known genes, was used as the control. The siCav1 and C‐siRNA were transfected into cells as described previously (Rao et al. 2006 (link), 2010 (link); Zhuang et al. 2013 (link)). Briefly, for each 60‐mm cell culture dish, 20 μL of the 5 μmol/L stock siCav1, or C‐siRNA was mixed with 500 μL of Opti‐MEM medium (Invitrogen). This mixture was added to a solution containing LipofectAMINE 2000 in 500 μL of Opti‐MEM. The solution was incubated for 20 min at room temperature and gently overlaid onto monolayers of cells in 3 mL of medium, and cells were harvested for various assays after 48‐h incubation.