Indomethacin

Hank’s Balanced Salts Solution was from Invitrogen (Carlsbad, CA). ELISA kits were from eBioscience (San Diego, CA) (IL1α, IL1β, TNFα, IL6), from Immunology Consultants Laboratory Inc. (Portland, OR) (albumin), from R&D Systems (Minneapolis, MN) (CCL20) and from PeproTech (Rocky Hill, NJ) (CXCL1, CSF2, CSF3). Antibodies for flow cytometry analysis were from eBioscience (San Diego, CA) (anti-mouse CD45 eF450, CD11c PE, CD24 FITC, CD11b APC, MHC-II I-A/E PerCP-eF710, CD103 FITC) and from BD Biosciences (San Jose, CA) (anti-mouse Siglec F-PE and Ly6G (clone 1A8)-PE). QuickIII staining kit for cytospins was obtained from Astral Diagnostics, NJ. Butylated hydroxytoluene and indomethacin were from Fisher Scientific. Percoll, collagenase XI, Trypsin inhibitor, DNase I, RBC lysis solution were from Sigma-Aldrich (St. Louis, MO). Nylon cell strainers (70 μm) were from BD Biosciences (San Jose, CA). Qiasol lysis reagent, RNeasy Mini Kits and Mouse Cytokines & Chemokines RT² Profiler PCR Array were from Qiagen (Valencia, CA). Paraformaldehyde was from Electron Microscopy Sciences (Hatfield, PA). XTT Cell Viability Kit was from Cell signaling.

To assess differentiation potential, 1 × 10⁵ adipose stromal cells were plated on six-well plates with DMEM and supplemented with 10% FBS and 1% antibiotic/antimycotic until confluent. For bone differentiation, media was supplemented with 0.01 M β-glycerol phosphate (MilliporeSigma, St. Louis, MO, USA, 50020) and 100 mM ascorbic acid (Sigma, A4544) and was replaced every 2–3 days. On day 21, cells were collected for RNA extraction or stained with alizarin red. Alizarin red stain was extracted and quantified by measuring absorbance at 405 nm (Bio Tek, Shoreline, WA, USA, 7091000). For adipocyte differentiation, media was supplemented with 0.5 µM dexamethasone (Sigma, D4902), 0.5 mM 3-isobutyl methyl xanthine (Sigma, I7018), 0.5 µg/mL insulin (Sigma, I5500), and 50 µM indomethacin (Fisher Scientific, AC458030050) for 7 days. After 7 days, media was supplemented with 0.5 µg/mL insulin (Sigma, I0516) and was replaced every 2–3 days. On day 21, cells were collected for RNA extraction or stained with oil red O. Oil red O stain was extracted and quantified by measuring absorbance at 510 nm.

Indomethacin was purchased
from Acros Organics (Fisher Scientific, Geel, Belgium). Hydroxypropylmethylcellulose
2910, 5 mPa s (HPMC E5) was purchased from DDP Specialty Electronic
Materials (DDP Speciality Electronic Materials Plaquemine). Poloxamer
407, poly(vinylpyrrolidone) (PVP) K30, and sodium lauryl sulfate (SLS)
were obtained from BASF (BASF Chemtrade GmbH, Burghernheim, Germany).
Docusate sodium salt (DOSS) was purchased from Merck KGaA (Darmstadt,
Germany). Purified water was freshly prepared using a Milli-Q integral
water purification system (Milli-Q Advantage A10; Merck Millipore,
Merck A/S, Hellerup, Denmark). Ethanol for analysis was obtained from
Merck KGaA (Merck KGaA, Darmstadt, Germany). Acetone, laboratory reagent
grade ≥99%, was purchased from Fisher Scientific (Fisher Scientific,
Loughborough, U.K.). All suspensions were filtered with a Durapore
0.22 μm PVDF Membrane (Merck Millipore Ltd., Cork, Ireland)
prior to analysis by X-ray powder diffraction (PXRD) and scanning
electron microscopy (SEM).

Quercetin (purity ≥ 99%) and fatty acid-free HSA (purity ≥ 96%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and indomethacin (purity ≥ 99%) was purchased from Acros Organics, (Geel, Belgium). All chemicals were used without further purification. Binding of Quercetin and indomethacin to HSA was studied using spectrofluorimetry, molecular docking, and molecular dynamics simulations.

hFPTs and ASC-F cells were seeded at a relative viable density of 1.5 × 10³ cells/cm² in multiple 12-well cell culture microplates (Falcon^®, USA) in CM-FBS or CM-HPL growth medium, respectively. Cultures were appropriately maintained in both incubation conditions (i.e., normoxia and hypoxia) until cell monolayers attained 80% confluency (i.e., 6 ± 2 days). Thereafter, the specific culture medium was replaced with a specific adipogenic induction medium, composed of high-glucose DMEM (Gibco™, USA) supplemented with 10% v/v FBS (Sigma-Aldrich^®, USA), 2 mM L-glutamine (Gibco™, USA), ITS 1 × (i.e., final concentrations of 10 mg/L insulin, 5.5 mg/L transferrin, and 6.7 µg/L selenious acid, Corning^®, USA), 1 µM dexamethasone (Acros Organics™, ThermoFischer Scientific, USA), 100 µM indomethacin (Acros Organics™, USA), and 100 µM IBMX (Alfa Aesar™, ThermoFisher Scientific, USA). The induction medium was exchanged twice weekly for a period of 14 days for the different cell types and in both incubation conditions. At the end of the induction period, the cells were fixed with a 4% formalin solution and appropriately stained with Oil Red-O (Sigma-Aldrich^®, USA) for revelation of lipid droplet accumulation. Following staining, assay microplates were photographed on an Olympus CX30 contrast phase microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).