Chetomin c9623

Recombinant HDGF protein was generated as previously described [12 (link)]. The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): chetomin (C9623), Bay 11–7082 (B5556), Ponceau S solution (P7170), and β-actin (A5441). The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): VEGF (sc-152), p-AKT (sc-33,437), AKT (sc-1619), p-IκB (sc-8404), p65 (sc-372), STAT3 (sc-482) and the nucleolin neutralizing antibody (sc-8031). Other antibodies were obtained as follows: p-STAT3 (4113; Cell Signaling Technology, Inc., Danvers, MA, USA), IκB (ab32518; Abcam plc., Cambridge, UK), and HIF-1α (NB100–479; Novus International Inc., St Louis, MO, USA).

3T3-L1 preadipocytes were obtained from ATCC (Manassas, VA, USA) and maintained in high glucose-DMEM (HyClone, Logan, UT, USA) supplemented with 10% bovine serum (BS, Gibco 16170, Thermo Fisher Scientific) in a humidified atmosphere of 5% CO₂ at 37 °C. For differentiation, confluent 3T3-L1 preadipocytes were treated with 1 µg/ml insulin (Sigma-Aldrich, St. Louis, MO, USA), 1 µM dexamethasone (Sigma-Aldrich), 0.5 mM IBMX (Sigma-Aldrich), and 10% fetal bovine serum (FBS, Gibco 16000, Thermo Fisher Scientific). After 2 days, the medium was replaced with medium containing 10% FBS and 1 µg/ml insulin and was thereafter replaced every 2 days with medium containing 10% FBS. After 6 days, differentiated 3T3-L1 cells were stained with Oil Red O (Sigma-Aldrich). Oil Red O staining was performed as described previously^{8 (link)}. Sodium dichloroacetate (DCA, 347795), sodium l-lactate (L7022), and chetomin (C9623) were purchased from Sigma-Aldrich. GSK2837808A (# HY-10031) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).