Mntbap chloride

This research protocol had been approved by the institutional review board Fukushima Medical University.
3T3-L1 pre-adipocytes and L6 myocytes were purchased from the American Type Cell Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), streptomycin, trypsin, fetal bovine serum (FBS), TRIzol reagent, lithium dodecyl sulfate (LDS) sample buffer, and sample reducing agent were supplied by Invitrogen Life Technologies (Carlsbad, CA, USA). The cell culture insert was obtained from BD Falcon (Franklin Lakes, NJ, USA) and RNeasy kit, from QIAGEN Inc. (Valencia, CA, USA). The Akt antibody and anti-phospho-specific Akt (Ser473 and Thr308) was purchased from Cell Signaling Technology (Boston, MA, USA) and anti-glucose transporter Glut1 antibody, from Abcam (Burlingame, CA, USA).
Polyvinylidene difluoride (PVDF) transfer membranes were procured from Millipore Corp. (Bedford, MA, USA). iScript cDNA synthesis kit and iQ SYBR Green Supermix were provided by Bio-Rad Laboratories (Richmond, CA, USA). [³H] 2-deoxyglucose (2-DOG) was purchased from PerkinElmer Inc. (Waltham, MA, USA). The antioxidant Mn-TBAP chloride was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA) and apocynin, by Abcam (Cambridge, UK). DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) cellular ROS detection assay kit was obtained from Abcam (Burlingame, CA, USA).

Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin-EDTA, penicillin-streptomycin and dimethyl sulfoxide (DMSO), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (M2128), Hoechst 33258 (B1155) and DCF (D6883) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All tissue culture-ware was from Nunc (Roskilde, Denmark). Proteinase inhibitors were from Roche (Madrid, Spain). ECL western blotting substrate was from Pierce (Thermo Fisher Scientific, Rockford, IL, USA). Novex Sharp Pre-Stained Protein Standard (LC5800) (T-3168) were from Invitrogen Life Technologies (Carlsbad, CA, USA). Sorafenib (BAY 43-9006, Nexavar) and Regorafenib (BAY 73-4506, Stivarga) are manufactured by Bayer. Cabozantinib and A-1331852 were purchased from MedChem Express (Monmouth Junction, NJ, USA). Buthionine sulfoximine, MnTBAP chloride and glutathione monoethyl ester were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Fibroblasts were treated with 5 µM MnTBAP chloride (Santa Cruz Biotechnology, Dallas, TX, USA, CAS 55266-18-7), 1 µM Baricitinib (Abcource Diagnostics GmbH, Munich, Germany, LY3009104, INCB028050) or with a combination of the two drugs at above indicated concentrations. The treatment period was 9 days, with the culture medium containing the drugs changed every other day.