Atcc cl 173

3T3-L1 cells (ATCC^® CL-173™) were purchased from ATCC® via LGC Standards GmbH (Wesel, Germany). HEK 293 cells were kindly provided by Prof. Wolfgang Graier (Department of Molecular Biology and Biochemistry, Medical University Graz, Austria). GSNO and DETA/NO were obtained from Enzo Life Science (Lausen, Switzerland). Information about antibodies is provided in Supplemental Table 1. Complete Protease Inhibitor^TM Cocktail and PhosSTOP^™ Phosphatase Inhibitor Cocktail were from Roche Life Science (Vienna, Austria). All other chemicals (unless otherwise indicated) were obtained from Sigma (Vienna, Austria).

3T3-L1 fibroblasts were purchased from ATCC (ATCC^® CL-173^™) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% P/S. Prior to stimulation, all the cells were starved with serum-free media. A group of cells was serum-starved for 12 h and then was stimulated with aldosterone (1 µM) at different time points in fresh serum-free media. Another group of cells was serum-starved for 1 h and then, under this condition, was pretreated with aldosterone (1 µM) and/or CMPD101 (3 µM) for additional 12 h, as previously done by us (Esposito et al., 2011 (link)). Then, the cells have been stimulated with insulin (100 nm) or ISO (10 µM) dissolved in fresh serum-free media. Control unstimulated cells were maintained in serum-free media.

We produced hResistin in a eukaryotic cell line [6 (link)]. Briefly, the pcDNA5/FRT/TOPO TA vector containing C-terminal FLAG-tagged hResistin cDNA was integrated into the genome of the Flp-In™ T-REx™ 293 cell line in a Flp recombinase-dependent manner (Invitrogen, Carlsbad, CA). Production of recombinant (r) hResistin in T-REx 293 cells was induced by 1 μg/mL tetracycline. hResistin protein then was purified from the cell culture medium by anti-FLAG M2 antibody agarose (Sigma, St. Louis, MO) column chromatography. To determine the purity of eluted hResistin proteins, SDS-PAGE were employed using 4–20% Criterio Tris-HCl protein gel (#3450033, Bio-Rad, Hercules, CA) and Coomassie (#1610436, Bio-Rad) staining. We then stimulated 3T3-L1 embryonic fibroblasts (ATCC® CL-173™, ATCC, Manassas, VA) with hResistin at different doses for 10 minutes and lysed the cells with Laemmli sample buffer. We analyzed the cell lysates by immunoblotting with anti-phospho-Akt [2 (link), 18 (link), 20 (link)] (#4060, Cell Signaling Technology, Danvers, MA) and anti-GAPDH (G8795, Sigma-Aldrich) to determine the activity of the purified hResistin. Western blot analysis was performed as previously described [18 (link)]. The Trans-Blot Turbo Nitrocellulose Transfer Kit (#1704271, Bia-Rad) were used and protein bands were visualized by chemiluminescence (ECL; RPN2106, GE Healthcare, Marlborough, MA).

African green monkey renal epithelial cells (Vero) [ATCC CCL-81], human embryonic kidney cells (HEK-293) [ATCC CRL-1573], mouse fibroblast cells (3T3) [ATCC CL-173], and rat liver epithelial cells (BRL-3A) [ATCC CRL-1442] were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in DMEM, supplemented with 10% FBS, 100 IU/mL of sodium penicillin, and 40 μg/mL of gentamicin sulfate in a humidified atmosphere containing 5% CO₂ at 37 °C. These cell lines were selected on the basis of their susceptibility and wide use to evaluate cytotoxicity induced by chemical components [22 (link)].