Nsc74859

C57/BL6 male mice were purchased from Shanghai Laboratory Animal Company (SLAC, Shanghai, China). HNF1a−/− mice were obtained from Charles River Laboratories and heterozygous mice were mated to obtain homozygous mutant mice as reported [22 (link)]; 8-week-old male mice were fed with either NC (protein 18.3%, fat 10.2%, carbohydrates 71.5%, D12450B, Research Diets) or an HFD (protein 18.1%, fat 61.6%, carbohydrates 20.3%, D12492, Research Diets) ad libitum for up to 8 weeks. Started at the 5th week, indicated groups of mice were given NSC 74859 (Selleck), 5 mg/kg, i.v. every 2 days for 5 doses. All animal procedures were approved by the Institutional Animal Care and Use Committee at People's Hospital of Shanghai Putuo, Tongji University School of Medicine. Mice were sacrificed at the 8th week, and the livers were taken for weight, Oil red O staining, or further analysis. Serum was collected for biochemical assays.

Human hepatic HepaRG cells were seeded at low density in William’s E medium with GlutaMAX (Gibco), supplemented with 10% FBS (Hyclone II GE), 1% penicillin/streptomycin (Sigma), 5 µg/mL insulin (Sigma), 0.5 µM hydrocortisone hemisuccinate (Sigma). After 1 week at confluence, cells were shifted into the same medium supplemented with 50 µM hydrocortisone hemisuccinate and 2% DMSO (Sigma) for 2 more weeks to obtain confluent differentiated cultures. Cells were then treated with different drugs (Supplementary Fig. 1c) for the indicated time at the following final concentrations: sodium oleate 250 μM (Sigma); S3I-201 100 μM alias NSC74859 (Selleckchem.); N-acetylcisteina 10 mM (Sigma); Ruxolitinib 1 μM (Selleckchem.). Cytoxicity of compounds was tested by MTT assay (Supplementary methods and Supplementary Fig. 1d).

The HO-1 enhancer hemin was obtained from Sigma (St Louis, MO, USA). HO-1 inhibitor ZnPPIX was purchased from Cayman Chemical (Ann Arbor, MI, USA). Bortezomib, Trametinib (an ERK inhibitor) and NSC74859 (a STAT3 inhibitor) were obtained from Selleck Chemicals (Houston, TX, USA). Recombinant human Gas6 (R&D Systems), anti-Gas6 antibody (Bioworld Technology), Gas6 primary antibody (Solarbio Life Science) and mouse IgG isotype control antibody (SouthernBiotech) were used. The following primary antibodies were purchased from Cell Signaling Technology: HO-1, β-actin, total extracellular signal-regulated kinase (ERK), p-ERK, total signal transducer and activator of transcription 3 (STAT3), p-STAT3, caspase-3 and Bcl-2.

Cynandione A was extracted, isolated, and purified by the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, with ≥98% purity determined by ¹H-NMR and HPLC. The rabbit β-endorphin antiserum and recombinant rat IL-10 antibody were purchased from Abcam (Cambridge, United Kingdom) and R&D systems (Minneapolis, MN, United States) respectively. The adenylate cyclase inhibitor 2,5-dideoxyadenosine (DDA) and PKA activation inhibitor H-89 were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, United States), while the p38 activation inhibitor SB203580, CREB activation inhibitor KG501, and STAT3 activation inhibitor NSC74859 were purchased from Selleck Chemicals (Houston, TX, United States), Sigma-Aldrich (St. Louis, MO, United States), and Medchem Express (Boston, United States), respectively. The α7 nAChR antagonist methyllycaconitine citrate was purchased from APEx BIO (Houston, United States). Cynandione A was dissolved in 10% dimethyl sulfoxide (DMSO) and 20% polyethylene glycol (PEG400) in 0.9% normal saline for intrathecal injection and dissolved in 0.1% DMSO for cell culture. All other drugs or reagents were dissolved or diluted in normal saline.