Pro caspase 3

Total protein was extracted from cultured cells using radioimmunoprecipitation (RIPA) lysis solution and protein concentration was assessed. Equal amount of proteins were separated by SDS-PAGE, and then transferred onto PVDF membranes. After being blocked by 5% non-fat milk in 0.1%TBST (Tris-buffered saline), the membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with HRP-conjugated secondary antibodies for 1 h. Protein signal was detected using an enhanced chemiluminescence (ECL) kit. GAPDH was used as an internal control to normalize protein levels. The primary antibodies used in this study include Bcl-2 (Abcam, Cambridge, MA, USA), BAX (Abcam), Pro-caspase-3 (Abcam), cleaved-caspase-3 (Abcam), ACAN (Santa Cruz, USA), COL2A (Santa Cruz), MMP13 (Santa Cruz), LC3 (Proteintech, Chicago, IL, USA), Beclin-1 (Proteintech), p-62 (Proteintech) and GAPDH (Santa Cruz).

Human esophageal squamous carcinoma cells, TE-1, were cultured in RPMI1640 supplemented with 10% fetal bovine serum and 100 mg/l streptomycin (Sigma) at 37°C in an atmosphere containing 5% CO₂. The reagents used in the present study were DCA, 3-MA (both Sigma-Aldrich, St. Louis, MO, USA), adenovirus (GFP-RFP-LC3; Hanbio Shanghai, China), Lipofectamine 2000 (Invitrogen Life Technologies, Sanghai, China), and an MTT kit (Sangon Biotech, Sanghai, China). The antibodies used in the present study were Beclin-1 (Cell Signaling Technology, Inc., MA, USA), LC3-I/II, P62, Atg5, PARP, Pro-caspase-3 (all Abcam, Cambridge, UK), β-actin (Santa Cruz Biotechnology Inc., Dallas, TX USA), Akt, phospho-Akt, mTOR and p-mTOR (all Cell Signaling Technology,).

Following treatment with propofol, total protein was isolated from KGN cells using RIPA lysis solution (Beyotime Institute of Biotechnology). The BCA protein quantitative kit (Beijing Solarbio Science & Technology Co., Ltd.) was employed to measure the protein concentration in accordance with the manufacturer's instructions. Subsequently, the extracted protein samples (40 µg per lane) were loaded on 10% SDS-PAGE and transferred on PVDF membranes. The membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with the following primary antibodies: Wnt3a (cat no. ab219412; Abcam), β-catenin (cat no. ab16051; Abcam), cleaved caspase3 (cat no. ab32042; Abcam), pro-caspase3 (cat no. ab32499; Abcam) and GAPDH (cat no. ab9485; Abcam) at a dilution of 1:1,000 overnight at 4˚C. The following morning, the membranes were incubated with the corresponding secondary antibody (cat no. ab7090; 1:2,000; Abcam) for 1 h at room temperature. The protein bands were exposed using EasyBlot ECL Kit (Shanghai BestBio) and analyzed using Image J (National Institutes of Health).

Cells (48 h after transfection) were washed once with PBS and lysed in RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.). Protein samples were quantified with the Pierce BCA Protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) and were then boiled for 10 min in sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of protein (20 µg/well) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. After blocking with 5% skim milk in 0.05% TBS-Tween-20 (v/v) for 1 h at room temperature, the membranes were incubated with the appropriate primary antibodies (at a dilution of 1:1,000) overnight at 4°C. Antibody specific to actin (C-2; cat no. 4967) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); c-Myc (cat no. ab32072), active caspase-3 (cat no. ab2302), p27 (cat no. ab54563), p21 (cat no. ab7960) and pro-caspase-3 (cat no. ab32150) were purchased from Abcam. Bax antibody (cat no. 2772) was purchased from Cell Signaling Technology, Inc. Then membranes were washed once with TBS-Tween-20 and incubated with anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies (cat nos. ab131368 and ab191866, respectively; 1:5,000) for 2 h at room temperature. Protein bands were visualized by using WEST-ZOL (plus) Western Blot Detection system (Intron Biotechnology, Inc., Seongnam, Korea).