Mitochondria and PS 341 cytosolic protein extracts were prepared using a Mitochondria Isolation Kit (Pierce) according to the manufacturer’s instructions. Isolated mitochondria were solubilized in

a lysis buffer containing 20mM Tris–HCl (pH 7.5), 1% NP-40, 150mM NaCl, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2mM MgCl2, 1mM ethylene glycol tetraacetic acid (EGTA), 50mM β-glycerol phosphate, 25mM NaF, 1mM DTT, 1mM Na3VO4 with 2 mg/mL leupeptin, 2 mg/mL pepstatin A, 2 mg/mL antipain, and 1mM phenylmethylsulfonyl fluoride (PMSF). The mitochondrial proteins were then subjected to immunoblotting analysis using antibodies against Bax and Bak. The cytosolic proteins were subjected to immunoblotting analysis using antibody against cytochrome Selleckchem BMS387032 c. The treated cells were washed with

ice-cold PBS and solubilized in a lysis buffer containing 20mM Tris with a pH of 7.5, 2mM MgCl2, 1mM DTT, 0.5% Triton X-100, 1mM EGTA, 25mM NaF, 1mM Na3VO4, 50mM ®-glycerol phosphate, 2 mg/mL leupeptin, 2 mg/mL pepstatin A, 2 mg/mL antipain, and 1mM PMSF. After incubating on ice for 1 h, the insoluble materials were removed by centrifugation at 14,000 × g for 15 min. 50 μg of protein from each sample was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), followed by electrotransfer onto a PVDF membrane (Millipore). The membrane was blocked with 5% nonfat milk in PBS with 0.1% Tween 20 and probed with the antibodies. The blots were washed and incubated with a horseradish peroxidase-coupled antimouse immunoglobulin G (IgG) or an antirabbit IgG antibody (Pierce) followed by detection with an electrogenerated chemiluminescence (ECL) revelation system (Bio-Rad). All values are performed in triplicate and expressed as mean ± standard deviation with Microsoft Office 2013 and imaged with Sigmaplot 10 (Systat Software Inc, San Jose, CA, USA). A Student t test was used for quantitative analysis, and the significant Etofibrate difference is shown as * p < 0.05, **p < 0.01, and ***p < 0.001. To determine the types of ginsenoside in SG, we analyzed MeOH extract of SG by an analytical high-performance

liquid chromatography. As shown in Fig. 1, the amount of four main ginsenosides in the total ginsenosides were 20(S)-Rg3 (11.33%), 20(R)-Rg3 (6.88%), Rk1 (16.72%), and Rg5 (11.97%). As shown in Fig. 1, the amount of ginsenoside Rg3, Rg5, and RK1 reached 50% of total ginsenosides in SG. A number of studies showed that (20S) ginsenoside Rg3, Rg5, and RK1 inhibit cell viability in various human cancer cells. We then examined whether SG features cytotoxic activity in human cancer cells in human cervical adenocarcinoma HeLa cells, human colon cancer SW111C cells, and SW480 cells through an MTT assay. Fig. 2 illustrates that SG exhibited a moderate cytotoxicity against the HeLa, SW111C, and SW480 cells with IC50 values of 94 μg/mL, 78 μg/mL, and 224 μg/mL, respectively.