Indomethacine

After expansion, MSC were subjected to adipogenic induction medium consisting of DMEM high glucose, 10% FCS, 1 μm dexamethasone, 0.2 mM indomethacine, 0.5 mM isobutylmethylexanthine (all Sigma, Deisenhofen, Germany), 0.01 mg/ml insulin (Sanofi-Aventis, Frankfurt, Germany), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured for 3 weeks with medium changed twice per week. To monitor adipogenesis, cells were fixed with 4% paraformaldehyde and stained with 0.3% Oil Red O solution (Chroma, Münster, Germany). Dye was re-extracted from vacuoles by 60% isopropanol and quantified by measuring its optical density at 490 nm.

Adipose-Derived human Mesenchymal Stem Cells (AD-hMSCs, Lifeline Cell Technology, Frederick, MD, USA) were expanded at 37°C and 5% CO2 in Mesenchimal Stem Cell Growth Medium 2, supplemented with Mesenchymal Stem Cell Growth Medium 2 Supplement Mix (PromoCell, Heidelberg, Germany). For differentiation experiments, confluent cells were switched to Mesenchymal Stem Cell Growth Medium 2 supplemented with 10% Foetal Bovine Serum (Biowest #S1810-500, Nuaillé, France), 0.2 μΜ Indomethacine (Sigma-Aldrich, #I7378), 10 μg/ml insulin (Sigma-Aldrich #I2643), 1 μM dexamethasone (Sigma-Aldrich #D2915), and 0.5 mM Isobutylmethylxanthine (Sigma-Aldrich, #I7018), to induce the differentiation into adipocytes. Medium was replenished every three days. For cell growth measurements, cells were detached with TrypLE Express (Gibco, #12304-021, Thermofisher) and were counted with a Cell Countess II FL (Thermo-Fisher Scientific).

The preparation of methanolic H. stoechas extract was previously described [12 (link)], and a plant voucher was deposited at the Universidad San Jorge herbarium (ref. 002–2014). The phenolic profile of this extract was previously determined [12 (link)] (see Introduction).
The compositions of the buffers used were as follows: Krebs buffer (in millimolar): NaCl 120, KCl 4.7, CaCl₂ 2.4, MgSO₄ 1.2, NaHCO₃ 24.5, KH₂PO₄ 1, and glucose 5.6. Ca²⁺-free Krebs: NaCl 120, KCl 4.7, CaCl₂ 2.40, MgSO₄ 1.2, NaHCO₃ 24.5, KH₂PO₄ 1, glucose 5.6, and ethyleneglycoltetraacetic acid (EGTA) 1. Ca²⁺-free high K⁺ Krebs ([K⁺]_o = 50 mM). All buffers were adjusted to pH 7.4. Apamin (AP), arzanol, barium chloride dihydrate (BaCl₂), glibenclamide (Glib), heparin (H), H-89 dihydrochloride hydrate, indomethacine, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), phenylephrine (PE), ruthenium red (RR) and verapamil (V) were obtained from Sigma (Madrid, Spain).
Tetraethylammonium (TEA), 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) and Rp-8-Br-PET-cGMPS were purchased from Tocris (Madrid, Spain). AP was diluted in acetic acid. Glib, TRAM-34 and arzanol were dissolved in dimethyl sulfoxide (DMSO). All other chemicals were dissolved in distilled water.

Dulbecco’s Modified Eagle’s Medium, alpha Modified Eagle’s Medium, Cosmic Calf Serum and Fetal Bovine Serum (FBS) were from Life Technologies. GW4064 was from Tocris. Ascorbic acid, β-glycerophosphate, bovine insulin, IBMX, dexamethasone, indomethacine, pioglitazone and rosiglitazone were from Sigma.

First-passage ASCs were replated on thermanox cover slips (Nunc, Roskilde, Denmark) for adipogenic differentiation, and another part for osteogenic differentiation. Adipogenic differentiation was performed using basic culture medium with addition of dexamethasone, insuline, indomethacine and 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St Louis, MO, USA). Differentiation was confirmed by Oil Red O stain. Osteogenic differentiation was performed using basic culture medium with addition of dexamethasone, ascorbic acid and β-glycerophosphate (Sigma-Aldrich, St Louis, MO, USA). Differentiation was confirmed by Von Kossa's Stain. Digital imaging of all wells was performed using an Olympus inverted microscope, and Cell^M software (Olympus Europe, Hamburg, Germany).

Celecoxib was purchased from Astatech Inc. (Bristol, PA, USA), while cinnarizine, JNJ-2A, ibuprofen, and itraconazole were obtained from Janssen Pharmaceutica (Beerse, Belgium). Fenofibrate and indomethacine were purchased from Sigma-Aldrich (Wicklow, Ireland). Progesterone, felodipine, sulfalazine, haloperidol, danazol, naproxen, venetoclax, carvedilol, dipyridamole, niclosamide, griseofulvin, fenofibric acid, ketoconazole, and clotrimazole were purchased from Kemprotec (Carnforth, UK), and Capmul MCM C8 was kindly donated by Abitec (Columbus, OH, USA). Maisine CC was a kind gift from Gattefossé (Lyon, France). All other chemicals and solvents were of analytical or high-performance liquid chromatography (HPLC) grade, purchased from Sigma-Aldrich (Wicklow, Ireland).