Fl83b

For AAV particle transduction experiments, cells were plated in ViewPlate-96 Black (PerkinElmer^®, Waltham, MA, USA) at cell type specific densities to ensure no overgrowth. The cells were infected in 1/3 dilutions of 150,000 VG/cell to 5000 VG/cell. Successful transduction was analyzed via IncuCyte^® S3 Live-Cell Analysis System (Sartorius, Göttingen, Germany) and flow cytometry after three days. Cell lines 4T1, B16-F10, bEND.3, CT26-CL25, NIH3T3, Renca, Tramp C2, and FL8-3B were purchased from ATCC (Manassas, VA, USA). MC-38 cells were purchased from NIH/NCI (Bethesda, MD, USA).

FL83B, a hepatocyte cell line derived from a normal liver taken from 15 to 17 day old fetal mice, was purchased from ATCC (Manassas, VA). Cells were maintained in F‐12K medium containing 10% FBS supplemented with 1% penicillin/streptomycin at 37°C. Before treatments with various compounds, cells were washed with PBS, and the medium containing 2% FBS was replaced overnight. The purpose of using 2% FBS medium for the cell treatment was to reduce basal cellular activity and bring all cells to the phase of growth arrest thereby equalizing all cells into the same phase of cell cycle which enables pronounced effect following treatment with growth signals (Zetterberg and Larsson 1985; Van Rechem et al. 2010). It also provides more reproducible experimental conditions (Colzani et al. 2009). Recombinant mouse myostatin protein was obtained from R&D Systems (Minneapolis, MN), AdipoRon was purchased from Sigma‐Aldrich (St Louis, MO), human recombinant insulin is a product from MP Biomedicals (Santa Ana, CA).

Mouse hepatocytes (FL83B, #CRL-2390) were purchased from ATCC (Manassas, VA, USA) and cultured in Kaighn’s modification of Ham’s F-12 medium (F12K, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (HyClone Laboratories, Logan, UT, USA). The human proximal tubular epithelial cells (HK-2, #CRL-2190) were purchased from ATCC and maintained in Dulbecco’s modified Eagle medium (DMEM)/F12K medium (Thermo Fisher Scientific), supplemented with 10% FBS and 1% penicillin/streptomycin (HyClone Laboratories). RAW 264.7 macrophage cells were maintained in DMEM medium containing 4.5 g/l of glucose, supplemented with 10% FBS and 1% penicillin/streptomycin (HyClone Laboratories). Cells were maintained at 37 °C in a humidified incubator containing 5% CO₂ and 95% air, and stimulated by LPS (1 µg/ml) to mimic an endotoxemia model in vitro. GOLPH3 specific small interfering RNA (siRNA) and corresponding control siRNA were purchased from Bioneer (Daejeon, South Korea). For transfection, the cells were transfected using the Lipofectamine^TM RNAiMax reagent (Thermo Fisher Scientific) by following the manufacturer’s instructions.