Ab181591

PBS at 4 °C was used to wash the cells twice. Thereafter, protease inhibitors in cold RIPA buffer were used to lyse the cells. Protein concentration was determined using BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China). Total protein of the cells were denatured using 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) was used to block the membrane for 1 h at room temperature. The membranes were then incubated with the primary antibodies overnight at 4 °C (dilution ratio 1:2000). TBST was used to wash the membranes three times and they were subsequently incubated with secondary antibodies (anti-rabbit IgG or anti-mouse IgG) for 1 h at room temperature. TBST was once again used to wash the membranes three times. The targeted proteins were identified using the ECL (EMD Millipore, MA, USA) method. SKA3, CCNE2, CCNA2, CDK4, CDK2, P53, P53-pSer15, P53-pSer46 antibodies used for western blot in this research were purchased from Bioss (bs-7848R), Abcam (ab32147), Abcam (ab108357), Abcam (ab181591), Abcam (ab32103), Abcam (ab32389), CST #9284 and CST#2521.

Human ARPE-19 cell line was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). Phillyrin (batch number Z17A8X34077, purity > 98%) was a product of the China Food and Drug Administration (Beijing, China). Dulbecco's Modified Eagle's Medium/nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin, and streptomycin solutions were purchased from Corning (NY, USA). Trypsin (0.05%) and phosphate buffered saline (PBS) were produced by Gibco, Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT), ML385, N-acetylcysteine (NAC)m and H₂O₂ solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Fas (ab82419), cytochrome c (ab90529), cleaved caspase-3 (ab2302), cleaved caspase-9 (ab2324), NQO1 (ab80588), Keap1 (ab118285), Bcl-2 (ab185002), Nrf2 (ab62352), CDK2 (ab32147), cyclin A (ab33911), cyclin E (ab181591), Bax (ab53154), β-actin (ab8226), p21 (ab188224), Histone H3 (ab1791), COX IV (ab16056) p53 (ab241556), pro caspase-3, pro caspase-8, and pro caspase-9 were obtained from Abcam, Cambridge, (MA, USA). Antibodies against p-p53 (9286S), cleaved caspase-8 (9496S), cleaved PARP (5625S), and HO-1 (86806S) were purchased from Cell Signaling Technology, Beverly, (MA, USA).

After 48 h of transfection, cells were washed three times with cold PBS. Then proteins were extracted from cells on ice by whole cell lysate, and the concentration was assayed using BCA kit (Thermo Fisher Scientific, Waltham, MA, United States). 30 μg of the total extraction was separated through polyacrylamide gel electrophoresis (PAGE), and transferred onto the PVDF membranes (Amersham, United States) that were sequentially blocked in 5% skim milk for 1 h. Afterward, the membrane was incubated with primary rabbit polyclonal antibodies overnight at 4°C, followed by horseradish peroxidase (HRP)-labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1:2000, Abcam, Cambridge, United Kingdom) at room temperature for 1 h. Primary antibodies contained CCNA2 (ab181591, 1:2000, Abcam, Cambridge, United Kingdom), p53 (ab32389, 1:1000, Abcam, Cambridge, United Kingdom) and GAPDH (ab9485, 1:2500, Abcam, Cambridge, United Kingdom). PBST (PBS containing 0.1% Tween-20) was utilized to wash the membranes three times following each step. Protein bands were visualized by chemiluminescence apparatus (GE, United States) and then photographed.

For western blot analysis, cells or tumor tissue were lysed in lysis buffer from the Qproteome Mammalian Protein Prep Kit (37901, QIAGEN) with the addition of protease inhibitors after PBS washing. Protein concentrations were measured by a microplate reader (Molecular Devices, California, USA) using the BCA Protein Assay Kit (P0010S, Beyotime, China). Then equal amounts of protein were resolved on SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA) and incubated with primary antibodies against: CDK4 (1:5000, ab108357, Abcam), cyclin D1 (1:1000, 554180, BD Bioscience, USA), cyclin  A2 (1:2000, ab181591, Abcam), cyclin B1 (1:50000, ab32053, Abcam), P21 (1:5000, ab109520, Abcam), PD-L1 (1:500, ab205921, Abcam or 1:2000, PA5-28115, Thermo Fisher scientific) and β-actin (1:2000, ab8227, Abcam); Secondary antibodies were goat anti-rabbit HRP (1:10000, ab6721, Abcam) and goat anti-mouse HRP (1:5000, ab97023, Abcam). Immunoreactive polypeptides were detected by electrochemiluminescence (ECL) reagents (Cat#170-5061, Bio Rad) using ChemiDoc™ XRS + Imaging System (Bio-Rad). Western blot band intensity quantification was calculated using ImageJ.

Human endometrial cancer cell lines (Ishikawa and AN3CA) were purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Bei Jing, China). Ishikawa, AN3CA were cultured in DMEM (10% fetal bovine serum) at 37 °C, 5% CO2 condition. siCDC20 and siCCNA2 purchased from Guangzhou Ribobio Co., Ltd. and transfected into cells by Lipofectamine RNAiMAX. The target sequence of siCDC20 was 5′-GACCACTCCTAGCAAACCT-3′, the target sequence of siCCNA2 was 5′-GCTGTGAACTACATTGATA-3′. After 48 hours of transfection, western blot was used to detect the efficiency of gene knockdown. The western blot was performed as previously described^{43 (link)}. The primary antibodies were anti-CDC20 (1:1000, Abcam, ab26483), anti-CCNA2 (1:1000, Abcam, ab181591) and anti-β-actin (1:1000, Abcam, USA). After 48 hours of transfection, cell growth curve (Cell Counting Kit-8 assay) was used to evaluate the proliferation of each group of cells. The CCK-8 assay was performed as previously described^{44 (link)}.