Anti cleaved caspase 3

Anti-caspase-3, anti-cleaved caspase-3, Anti-Bcl-2 and anti-Bax were purchased from Beyotime Biotech Co., Ltd. (Shanghai, China). Anti-caspase-7, anti-cleaved-caspase-7, anti-caspase-8, anti-cleaved-caspase-8, anti-caspase-9, anti-cleaved-caspase-9, anti-PARP, anti-cleaved PARP, anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Cell Signaling Technology. Anti-β-actin was purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China).
H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO for 24 h. Cells were collected and lysed with the Cell Lysis Solution RIPA (Beijing ComWin Biotech Co., Ltd) for 30 min on ice. Samples were spun down (12,000 g for 15 min at 4 °C) to collect the supernatants and protein concentrations were measured by BCA Kit (Thermo Fisher Scientific, USA). Equal amount of protein in each sample was isolated by 12% SDS-PAGE and transferred to PVDF membranes (Biosharp, China). After blocking with TBST buffer contained 5% nonfat milk, membranes were incubated with corresponding primary antibodies and secondary antibodies conjugated to horseradish peroxidase (HRP), respectively. After washing with TBST, the target proteins were detected by ECL assay kit (Beyotime, China).

The fat tissue extracts were prepared using 500 μL lysis buffer (Beyotime, Beijing, China) per 20 mg fat tissue, for 30 min at 4°C. Following centrifugation at 12,000 × g for 10 min at 4°C, the supernatants were removed and their protein concentrations determined using the BCA method. Total protein extracts were separated by 12% SDS-PAGE and transferred to polyvinylidene membranes (60 V for 4 h). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) at RT for 2 h, and probed overnight with primary antibodies at 4°C (anti-BCL2, anti-BAX, anti-cleaved caspase-3, anti-P53, anti-TNFR: Beyotime, Shanghai, China; anti-caspase3: ABcam, UK; anti-tubulin: Santa Cruz, USA). After washing with TBST, the membranes were probed with a horseradish peroxidase-labeled secondary antibody (1:10,000, Santa Cruz, USA) at RT for 1.5 h. Blots were visualized with a chemiluminescence reagent (Millipore, MA, USA) using an imaging system (BioRad, CA, USA).

PD was purchased from Must Bio-Technology Co., Ltd. (Chengdu, China) and sorafenib was purchased from Selleckchem (Houston, TX, USA). Fetal bovine serum (FBS) was purchased from Bioind (Biological Industries, BeitHaEmek, Israel). The anti-FOXO3a, anti-FasL, anti-Bim, anti-TRAIL, anti-Akt and anti-Ubiquitin antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-p-Akt, anti-ERK and anti-p-ERK antibodies were obtained from Abcam (Shanghai, China). The anti-Caspase 3, anti-Cleaved Caspase 3, anti-Caspase 3 and anti-PARP antibodies were obtained from Beyotime Biotechnology, Inc. (Shanghai, China). The anti-CDK4, anti-CDK6 and anti-cyclin D antibodies were obtained from Boster Biological Technology, Inc. (Wuhan, China). The Z-VAD-FMK, MG132, pictilisib and MK2206 were purchased from Selleckchem (Houston, TX, USA). Lipofectamine™ 2000 was purchased from Invitrogen (Shanghai, China). The shRNA-FOXO3a plasmid, cDNA-Akt plasmid and cDNA-FOXO3a were purchased Shanghai GeneChem Co., Ltd (Shanghai, China).