Cytochrome c

Human melanoma cell lines, Sk-Mel-28, A375, IGR37, LU-1205 and MV3 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and they were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), penicillin (100 IU/ml), and streptomycin (100 μg/ml) in a humidified incubator with 5% CO₂ at 37°C. Human primary melanocytes were obtained Kanglang Company (Shanghai, China) and cultivated in melanocytes growth with 10% FBS. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) and chaetocin were purchased from Sigma-Aldrich Corp. (St. Louis, MO). chaetocin was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution which was diluted to the final concentration with culture medium. The final concentration of DMSO was kept under 0.1% throughout the following studies, and showed no effect on cell morphology and proliferation in this study. The primary antibodies recognizing Bax, Bcl-2, procaspase-9/-3, cleaved caspase-9/-3, cytochrome c, PCNA, Nrf2, SOD2, catalase, and β-actin were purchased from Abcam Company (Cambridge, UK).

The extraction of cell cytoplasmic and mitochondrial fractions were performed using the Mitochondria/Cytosol Fractionation Kit (BioVision, CA, USA) according to the manufacturer’s protocol. Total cell proteins were extracted by RIPA lysing buffer (Beyotime Institute of Biotechnology, China) and protein concentration was determined by the BCA Bradford protein assay kit (Beyotime Institute of Biotechnology, China). The extracted proteins were then mixed with loading buffer and boiled for 10 min. Equal amounts of proteins (40 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). After being blocked with 5% non-fat dry milk for 2 h, membranes were incubated overnight at 4 °C with primary antibodies against GAPDH (1:1000, Abcam, USA), VDAC (1:2000, abcam, USA), cytochrome C (1:500, Abcam, USA), Bcl-2 (1:500, Abcam, USA), Bax (1:800, Abcam, USA), cleaved caspase 3 (1:500, Abcam, USA), caspase 8 (1:500, Abcam, USA), cleaved caspase 9 (1:500, Abcam, USA), and then incubated with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, China) for 2 h at room temperature. Membranes were visualized by an enhanced chemiluminescence (ECL) kit (Thermo, USA) and quantified by densitometry using an image analyzer (BandScan, USA).

Cells were lysed in a cell lysis buffer to obtain the total cellular extracts. The Pierce bicinchoninic acid (BCA) Protein Assay Kit was employed to determine the content of total protein in lysis cellular extracts (14 (link)). The sample protein was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and BCA quantified the protein sample concentration. Finally, the amount used in 10% SDS-PAGE was 20 μg. A nitrocellulose membrane (Mini-PROTEAN Tetra Cell, Bio Rad, Hercules, CA) was used to blot the SDS-PAGE. The HRP-conjugated secondary Ab or FLA9000 (Fuji Film, Minato, Japan) protein was visualized by electrochemiluminescence (ECL) using ChemiDoc-It (UVP, Upland, CA). The strip densitometry was performed using ImageJ Freeware (http://rsbweb.nih.gov/ij/). Antibodies used for blotting were Bcl-xl, 1:1,000; Bcl-2, 1:500; caspase3, 1:1,000; caspase9, 1:500 (Proteintech Group, USA); cytochrome C, 1:1,000 (Abcam, UK); and PARP-1, 1:500 (Cell Signal, USA).