Dexamethasone d2915

Bone marrow cultures were performed using a modified protocol of [68 (link)]. Briefly, WT and Foxo3^-/- bone marrow cells were enriched for hematopoietic progenitors using the EasySep Mouse Hematopoietic Progenitor Cell Enrichment Kit (#19756 Stemcell Technologies) and expanded at < 2x10⁶ cells/ml in non-treated 24-well plates with erythroid expansion medium. Erythroid expansion media consists of Stem Span SFEM (StemCell Technologies) supplemented with 2 U/ml human recombinant EPO (Amgen), 100 ng/ml SCF (PreproTech), 40 ng/ml insulin-like growth factor–1 (PreproTech), 10⁻⁶ M dexamethasone (D2915; Sigma), 0.4% cholesterol mix (Gibco) and 1% penicillin/streptomycin (Gibco). Two days later, cells were washed twice with PBS and plated at a concentration <1x10⁶ cells/mL in erythroid differentiation medium consisting of IMDM supplemented with 2 U/ml EPO, 100 ng/ml SCF, 10% Serum replacement (Invitrogen), 5% Platelet-Derived Serum, glutamine, MTG (1.27 μl in 10 ml of 1:10 dilution) and 10% Protein-Free Hybridoma Media. Cells were incubated in maturation medium for 3 days, with additional media added at day 3.

Cell counting kit-8 (CCK-8, CCK-3000) from Dongin Biotech (Seoul, Korea), bakuchiol (CFN-99047) from ChemFaces (Wuhan, China), and ascorbic acid (A-4544), β-glycero-2-phosphate (G-9891) and dexamethasone (D-2915) from Sigma-Aldrich (St. Louis, MO, USA) were purchased. For odontogenic differentiation, dexamethasone, β-glycerol 2-phosphate and ascorbic acid were added to Minimum Essential Medium-α supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) to obtain final concentrations of 0.1, 10 and 50 μM, respectively.

Phorbol 12-myristate 13-acetate (PMA, P8139), lipopolysaccharides from E. coli O111:B4 (L2630), adenosine triphosphate (ATP, A2383), potassium chloride (KCl, P5405) MG132 (C2211), folic acid (F876), allopurinol (A8003), and dexamethasone (D2915) were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Recombinant human TNFα (210-TA), human M-CSF (216-MC), mouse IL-1β (401-ML), and mouse TNFα (410-MT) were purchased from R&D Systems (Minneapolis, MN, USA). Monosodium urate crystals (MSU crystals, tlrl-msu), Salmonella flagellin (FLS-ST, tlrl-stfla), and poly (dA:dT) (tlrl-patn) were obtained from InvivoGen (San Diego, CA, USA). Complete Freund’s adjuvant (CFA, 7001) and incomplete Freund’s adjuvant (IFA, 7002) were obtained from Chondrex (Redmond, WA, USA). Colchicine (AG-CN2-0048) was purchased from AdipoGen (San Diego, CA, USA). Recombinant mouse M-CSF (315-02) was purchased from PeproTech (Rocky Hill, NJ, USA). Recombinant human IL-1β (RIL1B1) and disuccinimidyl suberate (DSS, 21655) were purchased from Thermo Fisher Scientific (Cleveland, OH, USA). Human NCOA6 siRNA (sc-61401), control siRNA (sc-37007), and febuxostat (144060-53-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ON-TARGETplus mouse Ncoa6 siRNA (L-041129-00-0005) and ON-TARGETplus nontargeting control siRNA (D-001810-10-05) were purchased from Dharmacon (Lafayette, CO, USA).