Cleaved poly adp ribose polymerase parp

The histone deacetylase (HDAC) inhibitor SAHA was purchased from Selleckchem (Houston, TX, USA) and the MDM2 inhibitor RG7388 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). The primary antibodies against p53, p21, p27, aurora kinase-B (AURK-B), cell division cycle 25C (CDC25C), cyclin dependent kinase 1 (CDK1), Bax, Bak, and cleaved poly (ADP-ribose) polymerase (PARP) (1:1000) were purchased from Cell Signaling Technology, (Danvers, MA, USA). MDM2 antibody (1:500 mouse monoclonal) was purchased from Santa Cruz Biotechnology, (Dallas, TX, USA). β-actin (1:2000) was purchased from Sigma Aldrich (St. Louis, MO, USA). The secondary antibodies anti-rabbit, anti-mouse, and horseradish-peroxidase (HRP) conjugated and dimethylsulfoxide (DMSO) were purchased from Sigma Aldrich. Nitrocellulose membrane (0.45 µm) was purchased from Amersham (GE Healthcare Life Sciences, Marlborough, MA, USA). ECL was purchased from KPL Biosolutions (Milford, MA, USA). DEVD-amc CellEvent^TM Caspase-3/7 Green ReadyProbe^TM was purchased from Thermo Fisher (Molecular Probes, Life Technologies, Carlsbad, CA, USA). Other chemicals and reagents used in this experiment were of research grade.

To analyze apoptosis, cells were trypsinized and plated into a six-well plate, and after 24 h, treated with EHGV (15.62 and 31.25 μg/mL) and CDDP (10 μM) for 48 h. Then, cells were lysed in 2× radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined using the DC Protein Assay kit (BioRad, Hercules, CA, USA). Equivalent amounts of protein (50 μg) were separated by SDS-PAGE. The gels were then transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk. Membranes were then incubated with primary antibodies against cleaved poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Danvers, MA, USA). Before blocking, the membranes were incubated with 1% glutaraldehyde for 30 min for the analysis of cleaved caspase-3 expression [30 (link)]. Detection was visualized with the ECL prime reagent (GE Healthcare, Sao Paulo, Brazil) and the images were then captured on a ChemiDoc Imaging system (Biorad, Hercules, CA, USA) using Image Lab software (Biorad, Hercules, CA, USA).

BA was supplied by MedChemExpress, USA. CCK-8 kit, dorsomorphin, AICAR, CQ, and Rapamycin (Rap) were provided by Selleck Chemicals, USA. Hoechst 33342 was procured from Sigma, USA. RiboBio Co., China, supplied ATG7 siRNA (SIGS0005319-1). NAC (N-acetyl-L-cysteine) was provided by Beyotime Biotechnology, China. Z-VAD-FMK powder was provided by Feifan Wang. The apoptosis detection kit (Cat. 556547) was purchased from BD Biosciences, USA. Antibodies against Bmi-1 (ab126783), Bcl-2 (ab182858), mTOR (ab2732), p-mTOR (Ser 2448), Bax (ab32503), AMPKα (ab32047), and LC3B (ab192890) were purchased from Abcam, UK. Anti-β-actin antibody (20536-1-AP) was supplied by Proteintech, USA. Antibodies against p-ULK1 (Ser555; Cat. 5869), cleaved poly (ADP-ribose) polymerase (PARP) (Cat. 9541), p-AMPKα (Cat. 2535), SQSTM1/p62 (Cat. 8025), and cleaved caspase 3 (Cat. 9664) were procured from Cell Signaling Technology, USA.

The treated cells were lysed with RIPA buffer containing 1% protease inhibitor cocktails and 2% phenylmethanesulfonyl fluoride (PMSF) (all from Sigma-Aldrich). For the detection of ERK translocation, the preparation of cytoplasmic and nuclear extracts was carried out as described in a previous study by Yang et al (21 (link)). After the supernatant collection, the protein concentration was determined using the Bradford method. Proteins were separated on a 10% SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (Bio Basic, Inc., Markham, Ontario, Canada). The transferred membranes were then blotted with primary antibodies (dilution of 1:1,000) to: phosphorylated ERK (p-ERKs), total ERK (t-ERKs), phosphorylated AKT (p-AKT), total AKT (t-AKT), cleaved poly(ADP-ribose) polymerase (PARP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lamin B (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight, followed by treatment with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Chemiluminescence was detected using ECL detection kits (GE Healthcare, Buckinghamshire, UK). The intensity of the bands was quantified by scanning densitometry using Quantity One 4.5.0 software (Bio Basic, Inc.).