Rosiglitazone rgz

HT (ref. 70604, CAS N°10597-60-1) and rosiglitazone (RGZ) (ref. 71740, CAS N°122320-73-4) were purchased from Cayman chemicals (Ann Arbor, MI). CA (ref. C0625, CAS N°331-39-5), sesame oil (ref. S3547), DMSO (ref. D8418), and ethyl 3-aminobenzoate methanesulfonate (MS-222) (ref. E10521) were provided by Sigma-Aldrich (Tres Cantos, Spain). Certified analytical grade ATX (ref. DRE-CA10307000, CAS N°472-61-7) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Stock solutions were stored at -20°C and working solutions were diluted in 0.1% DMSO on the day of the experiment.

The following drugs were used: T0070907 (T007;
2-Chloro-5-nitro-N-4-pyridinylbenzamide; Tocris, Cayman
Chemical, Michigan, USA); rosiglitazone (RGZ; Cayman Chemical, Michigan, USA);
Saquinavir, and Raltegravir (NIH AIDS Reagent Program, Maryland, USA).

The 3T3-L1 preadipocyte cell line (American Type Culture Collection, Manassas, VA) was grown in sterile 75 cm² polystyrene, cell+ growth surface flasks equipped with ventilated screw caps (Sarstedt, Nümbrecht, Germany) in basal medium (DMEM/GlutaMAX high glucose plus sodium pyruvate [Fisher Scientific, Vienna, Austria]) supplemented with 10% fetal bovine serum (Fisher Scientific) and 1% penicillin/streptomycin (Sigma, Vienna, Austria). Cells were split when reaching about 90% confluence with 1× trypsin-EDTA solution (Sigma) and were transferred to collagen-coated 6-well or 96-well plates, which were coated with collagen type I (rat tail; Fisher Scientific, 80 μg/ml working solution in 20 mM acetic acid) overnight at 4°C and afterward washed once with 1× PBS.
For adipocyte differentiation, 5-day postconfluent 3T3-L1 cells were exposed for 3 days to the differentiation medium 1 consisting of basal medium supplemented with 34.4 nM insulin (Sigma), 0.25 μM dexamethasone (DEX) (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma), and 2 μM rosiglitazone (RGZ) (Cayman, Tallinn, Estonia). On day 4, the medium was changed to differentiation medium 2 (basal medium supplemented with 34.4 nM insulin only) for the rest of the differentiation protocol until day 11.