Cox 4

Human laryngeal carcinoma tissues or cells were washed with ice-cold PBS and lysed with RIPA lysis buffer (Beyotime, Jiangsu, P.R. China) containing protease and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN, USA) at 4°C. Mitochondrial fractions were extracted using the Mitochondria Isolation Kit (Sigma-Aldrich) according to the supplier’s instructions. The protein concentration was determined by Enhanced BCA Protein Assay Kit (Beyotime). An equal amount of protein was separated by 10%–12% SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Five percent nonfat milk was added to membranes to block nonspecific binding at room temperature for 1 h. The membranes were then incubated with the following primary antibodies: TIMP-3 (1:500), Cox-IV, GAPDH, β-actin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); cytochrome c, caspase 9, and caspase 3 (1:1,000; Cell Signaling Technology, Billerica, MA, USA). The immunoreactive proteins were detected with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit or rabbit anti-mouse; 1:1,000; Beyotime) and an enhanced chemiluminescence reagent (Pierce Biotech, Rockford, IL, USA). Densitometry of bands was quantified using ImageJ software (NIH, Bethesda, MD, USA).

The SW used in this study was extracted from Oxytropis kansuensis Bunge and characterized in our laboratory according to the published protocol 22 . Its purity was up to 99%. SW was dissolved in phosphate buffered saline (PBS, 0.01 mol/L, pH 7.2) at 10 mg/mL as stock solution, and stored at -20 ^oC after sterile filtration.
Antibodies against caspase-8, caspase-9, caspase-3, PARP, Bid, Bcl-2, Bax, cytochrome c, COX IV and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Wuhan Boster Bio-Engineering (Wuhan, China). Caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD-fmk) and caspase-3 inhibitor (z-DEVD-fmk) were all purchased from Sigma-Aldrich (St. Louis, MO, US). TUNEL BrightRed Apoptosis Detection Kit was purchased from Vazyme (NJ, USA). All of other chemicals and reagents were the highest quality and obtained from standard commercial sources.
Primary GTCs were obtained according to our previous study 23 (link), and cultured in complete DMEM/F12 culture medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin, at 37 ^oC in a 5% CO₂ atmosphere incubator. All animal experiments were carried out in accordance with policy and ethical guidelines.