Tenovin 6

RITA was purchased from Cayman Chemical (Ann Arbor, MI) and the protein kinase inhibitor staurosporine was purchased from Sigma (St Louis, MO). The SIRT1 inhibitor Tenovin-6 was obtained from Santa Cruz Biotechnology (Dallas, TX). RITA was dissolved in 100% dimethylsulfoxide (DMSO) to a stock concentration of 50 mM and stored as aliquots until use. staurosporine and Tenovin-6 were dissolved in 100% DMSO and water, respectively, to stock concentrations of 1 M and stored as aliquots until use.

Commercially available Hep3B, RAW264.7 cells (ECACC, Sigma-Aldrich, St. Louis, MO, USA) were used. LX2 cells were kindly given by Dr. Ramón Bataller (IDIBAPS, Barcelona, Spain). All cell lines are mycoplasma-free. Human recombinant TNF (Peprotech, Rocky Hill, NJ, USA), LPS (E. coli 0111:B4, Sigma-Aldrich) were administered to cells at 50 ng/ml. CTSB inhibitor (CA-074 methyl ester, Sigma-Aldrich) was given at 25 μM to primary hepatocytes, HSCs, Hep3B and LX2 cells and at 75 μM to KCs and RAW264.7 cells. CTSS inhibitor (Z-FL-COCHO, Calbiochem, San Diego, CA, USA) was given at 10 μM to primary hepatocytes, HSCs, Hep3B and LX2 cells and at 7.5 μM to KCs. Resveratrol (100 μM) and Tenovin-6 (10 μM) (Santa Cruz Biotechnology, Dallas, TX, USA) were given to LX2 cells. Control and human SIRT1 shRNA lentiviral particles commercially available were used (Santa Cruz Biotechnology).

The cells were seeded in 6-well plates at 1 × 10⁵ cells/ml/well 1 day before the transfection. The following day the transfection was performed when the cells reached ~70% confluence. The final concentration of IL-17A or HMGB1 siRNA (Santa Cruz) was 100 nM. The transfection was conducted with the X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's instructions. The transfection medium was replaced 4–6 h after the transfection. After siRNA transfection, Tenovin-6 (10 μM; SantaCruz, USA) and MK-2206 (5 μM; Selleck, China) were added or not.