Antibodies against caspase 3 and caspase 9

Stock solutions of NCTD (Yuanye Bio-Technology Co., Ltd., Shanghai, China), 5-FU (Selleck Biotech Co., Ltd., Houston, TX, USA), and DMSO (Sigma-Aldrich, St. Louis, MO, USA) were prepared. The preparation of NCTD and 5-FU stock solutions: 20 mg NCTD powder was diluted to 100 mM with 1.2 mL DMSO. The working concentration was 12.5/25 μM. Analogously, 20 mg 5-FU powder was diluted to 100 mM with 1.5 mL DMSO. The working concentration of 5/10 μM was then prepared. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (PS), and trypsin were obtained from Gibco, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). β-actin monoclonal antibodies were purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Antibodies against caspase-3 and caspase-9 were purchased from Cell Signaling Technology Inc. (Boston, MA, USA). Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP were purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA). Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology (Shanghai, China). The Annexin V-FITC/PI Apoptosis Detection Kit and TUNEL FITC Apoptosis Detection Kit were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China).

Cells were lysed in RIPA lysis buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF) to obtain total protein. Protein extracts were electrophoresed on SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes followed by blocking with 5% non-fat dry milk in Tris-buffered saline and 0.1% Tween (TBST) at room temperature. The membranes were incubated overnight at 4°C with primary antibodies diluted in blocking solution. After washing in TBST, the corresponding horseradish peroxidase-conjugated secondary antibodies were added for 1 h. Protein bands were visualized by chemiluminescence using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, U.S.A.). Antibodies against β-actin, PARP1, p21, CDK2, Histone-H3, α-Tubulin and acetylated α-Tubulin were purchased from Proteintech. Antibodies against Caspase-3 and Caspase-9 were from Cell Signaling Technology. Antibodies against Histone H3Ac and Histone H4Ac were from Active Motif.