Tgf β 240 b

The TGF-β activity was measured based on its ability to induce plasminogen activator inhibitor-1 (PAI-1) expression, which was measured with a PAI-1 luciferase reporter assay (Addgene, Cambridge, MA, USA). The luciferase reporter plasmid containing PAI-1 binding sites was transfected into cells. After modulation of TGF-β or GDF15 expression by transfection of the specific shRNA or the addition of the recombinant proteins (TGF-β, #240-B or GDF15, #957-GD, R&D Systems, Minneapolis, MN), the luciferase activity in each experiment was determined as previously described [66 (link)]. All the experiments were performed triplicate for three times and the similar results were obtained. The error bars shown in the relevant figures indicated the standard deviation of a triplicate experiment.

AMP‐activated protein kinase (cat. no. 2532, 1 : 1000) and Thr‐172 AMPK antibodies (cat. no. 2535, 1 : 1000) were obtained from Cell Signaling Technology (Danvers, MA, USA). All horseradish peroxidase (HRP)‐linked secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). AICAR (cat. no. A9978) and α‐SMA (cat. no. A2547, 1 : 5000) were from Sigma‐Aldrich (St Louis, MO, USA). Lipofectamine 2000 (cat. no. 11668019), Bodipy 493/503 (cat. no. D3922), Lysotracker red (cat. no. L7528), ProLong Gold Antifade Reagent (cat. no. P36931) and Alexa Flour 633 coupled anti‐mouse secondary antibody (cat. no. A‐21052) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). TGF‐β (240‐B) was purchased from R&D Systems (Minneapolis, MN, USA). Unless indicated, other chemicals were obtained from Sigma‐Aldrich. The human LX‐2 cell line was a gift from S. Friedman (Mount Sinai School of Medicine) 18.

CAA was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, U.S.A.) to a concentration of 200 μM and stored at −20°C as a stock solution. The stock solution was diluted with Dulbecco’s modified Eagle’s medium (DMEM, HyClone, U.S.A) to a final concentration of 50 μM before use. Dexamethasone (Cell Signaling Technology, #9668) was diluted with DMEM to a final concentration of 1 μM. DMSO was added in the vehicle control group. TGF-β (240-B; R&D Systems, MN, U.S.A.) was diluted to a concentration of 6.4 ng/ml, and IL-6 (206-IL/CF, R&D Systems, MN, U.S.A.) was diluted to a concentration of 8 ng/ml before use. Insulin (Cell Signaling Technology, #9668), human IL-6 high sensitivity enzyme-linked immunosorbent assay (ELISA) Kit (Abcam, ab46042, U.S.A), human IL-17 High Sensitivity ELISA Kit (Abcam, ab46042, U.S.A), propidium iodide (Cell Signaling Technology, #9668), 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, #9668), primary antibodies including anti-IL-17RC, anti-IL-6, anti-p-IR (Cell Signaling Technology, #9542), anti-GLUT1 (Cell Signaling Technology, #9542), and monoclonal mouse anti-GAPDH (Santa Cruz, CA, U.S.A.), and horseradish peroxidase (HRP)–conjugated polyclonal goat anti-mouse and anti-rabbit (both from Santa Cruz, CA, U.S.A.) were used in the present study. Human IL-17RC adenovirus was purchased from Vector Biolabs (Malvern, PA, U.S.A.).