Cleaved caspase 9

RIPA protein extraction reagent (Beyotime, Shanghai, China) with the addition of PMSF (Roche, Basel, Switzerland) was used to lyse the cells. Protein extracts were separated by utilizing the 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and were then transferred onto the nitrocellulose membranes (Sigma, St. Louis, MO, USA), which were incubated with specific primary antibodies for 12 h, followed by 2-h incubation with secondary antibodies. Detection of protein bands was done by the enhanced chemiluminescent (ECL) method. Primary antibodies against total caspase 3, cleaved-caspase 3, total caspase 9, cleaved-caspase 9, BAZ2A, PCNA, Ki67, and GAPDH were all from Abcam (Cambridge, MA, USA).

We obtained Dulbecco's Modified Eagle Medium (DMEM) and 0.25% Trypsin from Thermo Scientific HyClone (Logan, USA). Fetal bovine serum (FBS) was purchased from Gibco (New York, USA). Dimethyl sulfoxide (DMSO) was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). Cell counting kit-8 (CCK-8) and Apoptosis Detection Kit were obtained from the Dojindo (Kumamoto, Japan). Cell Cycle Detection Kit was purchased from KeyGEN Biotech (Nanjing, China). We purchased anti-rabbit secondary antibodies and the primary antibodies against tubulin, cytochrome C (cytoplasm), B-cell lymphoma (Bcl-2), cleaved caspase 3, P53, Bid, Bax, cleaved caspase 9, cyclin-dependent kinase 2 (CDK2), cell division control protein 2 (CDC2), cleaved caspase 8, cleaved PARP, and cyclin B1 from Abcam (Cambridge, UK). We purchased primary antibodies against phosphorylated retinoblastoma protein (P-Rb), cyclin A2, cyclin E, and E2F1 from HUABIO Biotechnology (Hangzhou, China).
Caffeic acid, p-coumaric acid, CAPE, ferulic acid, isoferulic acid, kaempferol, 3,4-dimethoxycinnamic acid, pinocembrin, naringenin, quercetin, apigenin, chrysin, and galangin were obtained from Sigma-Aldrich (Missouri, USA). Pinobanksin, 3-O-acetyl pinobanksin, and benzyl caffeate were obtained from Haishu Apexocean Biochemicals (Ningbo, China), and benzyl p-coumarate was purchased from Kunming BioBioPha Co., Ltd. (Kunming, China).

According to the manufacturer’s instructions, a nuclear and cytoplasmic protein extraction kit (Beyotime) was used to extract total protein, cytoplasmic protein, and nuclear protein from NP cells and tissues. The BCA protein analysis kit (Beyotime) was used to determine the protein concentration. Equal amounts of protein from each sample were separated using SDS-PAGE and transferred to a PVDF membrane. Non-fat milk (5%) was used to block the membranes at room temperature for 2 h. Then, the membranes were incubated overnight at 4°C with the following primary antibodies: Bax (Abcam), Bcl-2 (Abcam), cleaved caspase-9 (Abcam), cleaved caspase-3 (Cell Signaling Technology), cytochrome c (Abcam), collagen II (Abcam), MMP-13 (Thermo Fisher), Nrf2 (Abcam), and HO-1 (Proteintech). Histone (Abcam) and GAPDH (Cell Signaling Technology) were used as the internal controls. Subsequently, the membrane was washed with TBST and incubated with the respective secondary antibodies (Abcam) for 1 h at room temperature, and then, the membranes were washed with TBST again. Protein bands were observed by enhanced chemiluminescence (Thermo Fisher) according to the manufacturer’s instructions. ImageJ software (NIH) was used to quantify band intensity.

Polyphyllin VI (batch no. 111592-201604, purity > 97.0%) was purchased from China Food and Drug Testing Institute, Beijing, China. RPMI 1640 medium, penicillin, streptomycin, and FBS were provided by Gibco-Life Technologies, Waltham, MA, USA. MTT was product of Biotopped Life Sciences Co. Ltd., Beijing, China. LDH assay kit, DAPI assay kit, NAC, Annexin V-FITC apoptosis assay kit, ROS assay kit, MMP assay kit (JC-1), cell cycle assay kit(PI) were purchased from Beyotime (Nanjing, China). Antibodies for Bax (1:1000; rabbit polyclonal; cat. no. 5023; CST), Bcl-2 (1:1000; mouse polyclonal; cat.no. 15071; CST), p53 (1:1000; mouse polyclonal; cat. no. 2524, CST), p21 (1:1000; rabbit polyclonal; cat. no. 2947; CST), cyclin A2 (1:1000; mouse polyclonal; cat. no. 4656; CST), CDK2 (1:1000; rabbit polyclonal; cat. no. 2546; CST), cleaved caspase-3 (1:1000; rabbit polyclonal; cat. no. Ab2302; Abcam), cleaved caspase-9 (1:1000; rabbit polyclonal; cat. no. Ab2013; Abcam), cleaved caspase-8 (1:1000; rabbit polyclonal; cat. no. Ab25901; Abcam), cytochrome c (1:1000; rabbit polyclonal; cat. no. 4280; CST) and PARP (1:1,000; rabbit polyclonal; cat. no. 9532; CST), β-Actin (1:1000; rabbit polyclonal; cat. no. 4280; CST) and PARP (1:1000; rabbit polyclonal; cat. no. AC001-M; Santa).