Fh535

DMEM culture medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY). The doxorubicin (DOX, catalog #ST1285), CCK8 (catalog #C0043), apoptosis detection kit (catalog #C1062L), TOPFlash (catalog #D2501) and FOPFlash (catalog #D2503) was purchased from Beyotime (Shanghai, China). Anti-human β-catenin (catalog #8480), cyclin D1 (catalog #55506), cyclin B1 (catalog # 12,231), survivin (catalog # 2808), c-Myc (catalog #18583), CDK2 (catalog #18048), p53 (catalog #2527), β-actin (catalog #4970) and Histone H3 (catalog #4499) antibodies were bought from Cell Signaling Technology (Beverly, MA, United States). FH535 (catalog #HY-15721) and BML-284 (catalog #HY-19987) was purchased from Med Chem Express (MCE, United States). Humantenidine, gelsemine, koumine, gelsenicin, gelsevirine, and sempervirine (HPLC≥98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The roots and stems of G. elegans were bought from a commercial source and authenticated by the Department of Pharmacognosy, School of Pharmacy, Fujian Medical University as previous reported (Liu Hao et al., 2008 ). Other chemicals were of analytical grade and commercially available.

To determine the effect of β-catenin on the proliferation and survival of PBMSCs, we randomly divided the cells into the following four groups according to different intervention methods: the no intervention group (serving as the control group), the β-catenin overexpression group (oeβ-catenin), the β-catenin knockdown group (shβ-catenin), and the β-catenin overexpression+β-catenin knockdown group (oeβ-catenin plus shβ-catenin). To determine the effect of Oct4 on the proliferation and survival of PBMSCs, we divided the cells into the no intervention group, the Oct4 overexpression group (oeOct4), the Oct4 knockdown group (shOct4), and the Oct4 overexpression+Oct4 knockdown group (oeOct4 plus shOct4). To evaluate the effects of β-catenin and Oct4 on cell growth and apoptosis, we added oeβ-catenin+shOct4 and shβ-catenin+oeOct4. After transfection, all groups of PBMSCs were cultured for 70 days for subsequent experiments to analyze the growth and apoptosis of PBMSCs.
To investigate the mechanisms of β-catenin-mediated cytoprotective effects against apoptotic cell death, we performed β-catenin activation in PBMSCs using oeβ-catenin or a β-catenin agonist (SKL2001, MedChemexpress, Monmouth Junction, NJ, USA), and β-catenin inhibition was carried out by shβ-catenin or a β-catenin inhibitor (FH535, MedChemexpress).

The HCC cell lines Huh7 and HepG2 were obtained from the China Center for Type Culture Collection (Wuhan, China). Both cell lines were cultured in Dulbecco’s Modified Eagle Medium (HyClone, USA) supplemented with 10% fetal bovine serum (Gibco). HCC cell lines were seeded into 96-well plates at 4000 cells per well(4 replicates). On the 4next day, the medium in the wells was replaced after diluting the drug in equal proportions and placed into a normoxic incubator (37°C, 5% CO2, and 21% O2) or anoxic incubator (37°C, 5% CO2 and 1% O2). AKT_inhibitor_VIII, FH535, BI_2536, and RO_3306 were purchased from MedChemExpress(USA). After 72 hours, the medium was changed to 10% Cell Counting Kit 8 solution and incubated for one hour, after which the absorbance at 450 nm was recorded using an ELx 800 Universal Microplate Reader(BIO-TEK, USA).