Naringenin nar

C2C12 myoblasts were seeded on culture plates in PM to achieve a 90% confluence, then the medium was changed to the basal differentiation medium (DM), supplemented with 20 µM epicatechin (EC, Sigma-Aldrich, Burlington, MA, USA), 10 µM genistein (GEN, Sigma-Aldrich), 10 µM luteolin (LUT, Sigma-Aldrich), 50 µM quercetin (QUE, Sigma-Aldrich), or 10 µM naringenin (NAR, Sigma-Aldrich) to induce differentiation. DM was DMEM containing 2% horse serum (HS; Thermo Scientific, Waltham, MA, USA) and 1% P/S. The differentiation lasted for 3 days, and the medium was changed every day.
PSCs were seeded on Matrigel-coated plates and cultured in PM for 2–3 days. When cells reached 90% confluence, differentiation was induced in DM supplemented with NAR at the indicated concentration for 5–7 days. In some experiments, ERβ antagonist PHTPP (MedChemExpress, Shanghai, China) at 10 μM was added to DM alone or in combination with NAR. The medium was changed every day.

Naringenin (Nar) (≥ 95%, N5893), streptozotocin (STZ) were purchased from Sigma (Sigma Aldrich, United States). Anti-PCNA (1:1000, ab29) and anti-OPN (1:500, ab63856) were purchased from Abcam. Anti-MMP2 (1:1000, bs-4605R) and anti-MMP9 (1:1000, bsm-54040R) were obtained from Bioss Biotechnology Co., LTD. (Beijing, China). Anti-α-SMA (1:1000, BM0002), anti-VEGFA (1:1000, BA0407) and anti-VEGFR2/KDR (1:1000, A00901-3) were purchased from Boster Biological Technology Co. Ltd. (Shanghai, China). Anti-CREB5 (1:1000, G420) was purchased from Santa cruz. The following antibodies: PI3K (1:1000, 4249), p-PI3K (1:500, 17366), Akt-pan (1:1000, 4685), p-Akt (Ser 473) (1:1000, 4060), Src (1:1000, 36D10), and p-Src (1:1000, D49G4) were obtained from CST. EdU Imaging kit was purchased from APE × Bio (United States). Trypsin, Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO.