Cytochrome c 4280

Human NSCLC cell HCC827, HCC1975, H1650, and H460 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). All cells were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO₂. All cells were maintained at the incubator according to the standard protocols and subjected to routinely checking for mycoplasma contamination. Antibodies against HK2 (#2867), Akt (#4691), p-Akt (#4060), cleaved-caspase 3 (#9664), Bax (#14796), VDAC1 (#4661), α-tubulin (#2144), cytochrome c (#4280), Akt1 (#2938), Akt2 (#2964), p-GSK3β (#5558), β-actin (#3700), anti-rabbit IgG HRP (#7074), and anti-mouse IgG HRP (#7076) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). The natural compound Erianin was from Selleck Chemicals (Houston, TX). Necrostatin-1, z-VAD-fmk, and 3-methyladenine were purchased from MedChemExpress (New Jersey, US). Lipofectamine 2000 transfection reagent for transient transfection was purchased from Thermo Fisher Scientific (Waltham, MA).

The cells were plated and treated as described above for 48 hours, and then were lysed in cell lysis buffer (Beyotime, P0013, Beijing, China) supplemented with 0.5 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime, ST506). The total cellular protein concentration was determined with a BCA Protein Assay Kit (Thermo Fisher Scientific Inc., 23227, Rockford, USA). The proteins were applied to sodium dodecyl polyacrylamide gel electrophoresis, and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Then membranes were blocked with 5% evaporated skimmed milk for 1 hour and probed overnight at 4°C with the following primary antibodies: cleaved-CASP 3 (9664), Cytochrome C (4280), Phospho-Chk1 (2348), Phospho-Chk2 (2197), Phospho-ATM (5883), Phospho-Histone H2A.X (9718), BAX (2772), Bcl-2 (2870), (all 1:1000; Cell Signaling Technology, Danvers, MA, USA), antibody against ACTB (1:2000; Zsbio, sc-53142, Beijing, China), followed by incubation with horseradish peroxidase coupled secondary anti-mouse (Zsbio, ZB-2305) or anti-rabbit antibodies (Zsbio, ZB-2301) for 1 hour at room temperature. The protein bands were visualized using ECL blotting detection reagents (Beyotime, P0018), and developed and fixed onto x ray films. ACTB was served as a loading control.