Anti caspase 3

Supercritical fluid (SCF) extract of Zanthoxylum bungeanum was purchased from Zhengzhou Xuemailong Food Spice Co., Ltd. in Henan Province, China. Both HCT-116 (human colon adenocarcinoma cell line), and HEK293T cells were obtained from the National Collection of Authenticated Cell Cultures in Shanghai, China. McCoy's 5A media, DMEM media, fetal bovine serum (FBS), and 0.25% trypsin solution were obtained from Invitrogen (Carlsbad, CA, USA). The Caspase 8 inhibitor Z-IETD-FMK and P53 inhibitor PFTα hydrobromide were obtained from MedChemExpress (Monmouth Junction, NJ, USA). Primary antibodies (anti-CDK4, anti-cyclin D1, anti-PCNA, anti-BAX, anti-Caspase 3, anti-Cleaved Caspase 3, anti-P53, anti-Bcl-2, anti-Caspase 9, anti-Cleaved Caspase 9, anti-Fas, anti-P21, anti-Caspase 8, anti-Cleaved Caspase 8, and anti-β-actin), anti-mouse, and anti-rabbit secondary were obtained from the Beyotime Institute of Biotechnology (Shanghai, China).

Following the previously established procedure^{24 (link)}, post 24 h naringenin treatment, the cells were lysed using RIPA buffer. Protein expressions of β-actin, MMP9, PI3K, AKT, and Caspase-3 were assessed through SDS-PAGE, transferred onto a PVDF membrane, the blots were cut prior to hybridization with antibodies, and cropped blots incubated overnight at 4 °C with primary antibodies including anti-MMP9 (Beyotime, 1:1000), anti-mTOR (Beyotime, 1:1000), anti-PI3K (Cell signaling, 1:1000), anti-p-PI3K (Cell signaling, 1:1000), anti-AKT (Cell signaling, 1:1000), anti-p-AKT (Cell signaling, 1:1000), anti-EGFR (ABclonal, 1:1000), anti-Caspase-3 (Beyotime, 1:1000), and anti-β-actin (Cell signaling, 1:1000). Secondary antibodies used were either GAM (goat anti-mouse IgG) or GAR (goat anti-rabbit IgG) (ABclonal, 1:2000).

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).