Cleaved parp1

Cells were washed with phosphate-buffered saline (PBS) and harvested in radioimmunoprecipitation assay buffer (Beyotime, Haimen, China) supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Equal amounts of protein were loaded on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was probed with specific antibodies overnight at 4 °C and was then incubated with secondary antibodies at room temperature for 1 h. Each protein band was visualized with an ECL reagent (Millipore). Primary antibodies specific for the following proteins were used: Bax, Bcl-2, cleaved Caspase 9, cleaved PARP1, and Survivin (all from Proteintech Group, Wuhan, China), cleaved Caspase 3 (Affinity, Cincinnati, OH, USA), ANKH (ABclonal, Wuhan, China), AQP1, β-actin, MMP13, COL2A1, and ADAMTS-5 (all from Bioss, Beijing, China).

KYSE150 and KYSE450 cells were incubated with various concentrations of GA (0, 0.5 or 1 μM) for 24 hours, respectively. After washing twice with PBS, the cells were collected by trypsinization and centrifugation, and then subjected to a total protein extraction. Protein concentrations were measured by using Plyle BCA protein quantitation kit (Applygen, China). The equal amount of protein samples were separated by SDS-PAGE gel electrophoresis and transferred onto a PVDF membrane (Millipore, USA). The PVDF membrane was first blocked in 5% skim milk or 2% BSA and then incubated with primary antibodies, followed by the corresponding secondary antibodies (Abbkine, Wuhan, China). The protein targets in the membrane were detected and visualized by using the Chemiluminescence Luminol Kit. The following primary antibodies were applied in the experiments: cleaved-caspase3, cleaved-caspase9, cleaved-PARP1, phosphorylated AKT, Bcl-2, MMP2, MMP9 and β-actin (Proteintech Group, Wuhan, China); PARP1, BAX, PI3K, total AKT, phosphorylated AKT (ser473), p-mTOR (ser2448), cyclinB1 and cyclinD1 (Cell Signaling Technology, MA, USA). β-actin was chosen as a loading control in all experiments.

Reagents, antibodies and kits in the study were purchased as follows: GA from Sellck (Houston, USA), antibodies for cleaved-caspase 3, cleaved-caspase 9, cleaved-PARP1, Bcl-2, MMP2, MMP9 and β-actin from Proteintech (Wuhan, China) antibodies for Bax, PI3K, m-TOR, p-mTOR (ser2448), cyclinB1, cyclinD1, totalAKT and p-AKT (ser473) from Cell Signaling Technology (Danvers, MA, USA), FITC-conjugated Annexin V kit for apoptosis detection from NEOBIOSCINECE (Shenzhen, China), 4',6-diamidino-2-phenylindole (DAPI) from Beyotime (Shanghai, China).