Human recombinant tgf β1

Human proximal tubular (HK-2) cells were purchased from American Type Culture Collection (CRL-2190) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium which was contained 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere with 5% CO₂ at 37°C. For TGF-β1 stimulation, cells were treated with human recombinant TGF-β1 (5ng/mL; Santa Cruz, CA, USA) after being starved overnight, for 0 h, 24 h, 48 h, or 72 h. For siRNA-Klotho intervention, cells with the density of 1.0×10⁶ were starved overnight and transfected with siRNA-Klotho (siRNA-kl; 2μL) or siRNA-Negative Control (siRNA-NC; 2μL) for 24 h, which was constructed by GeneChem Biotech (Shanghai, China). The sequence of siRNA-Klotho was: GCAGAATTACATAAACGAA. Transfection of siRNA was carried out using Lipofectamine 3000 transfection reagent following the manufacturer’s instructions. After treatment of siRNA-Klotho, cells were treated with TGF-β1 (5 ng/mL) or control solutions for 48 h. To explore the effect of β-catenin in the anti-fibrotic procedures mediated by Klotho, ICG-001 (25 μM, Selleck Chemicals, USA), the specific inhibitor of β-catenin, was added with or without TGF-β1 to the HK2 cells.