Materials
Mouse insulinoma cell line MIN6 was kind gift from Dr. Makoto Shigeto (Division of Diabetes and Endocrinology Department of Medicine Kawasaki Medical School), Kohei Kaku (Division of Diabetes and Endocrinology Department of Internal Medicine Kawasaki Medical School), and Jun-ichi Miyazaki (Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine). Fetal bovine serum (FBS) was obtained from Thermo Fisher Scientific, Inc. (Kanagawa, Japan). Phosphate buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were obtained from Sigma-Aldrich, Inc. (St. Louis, MO). CoQ10 (oxidized form) was obtained from Yokohama Oils & Fats Industry (Kanagawa, Japan). Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) was obtained from Peptide Institute, Inc. (Osaka, Japan). STS and polyacrylamide gels were obtained from Wako Pure Chemical Industries (Osaka, Japan). A protease inhibitor cocktail was obtained from BioVision, Inc. (Milpitas, CA). Tris/Glycine/SDS electrophoresis buffer was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Western Blotting Detection Reagent was obtained from GE Healthcare (Tokyo, Japan). Polyvinylidene fluoride (PVDF) microporous membranes were obtained from Merck Millipore (Billerica, MA). Cell lysis buffer, an anti-β-actin antibody, anti-rabbit IgG, and annexin V assay kit were obtained from MBLCo., Ltd. (Aichi, Japan). An anti-caspase-3 antibody was obtained from Cell Signaling Technology (Tokyo, Japan). A cytochrome c releasing apoptosis assay kit was obtained from Funakoshi Co., Ltd. (Tokyo, Japan). An apoptotic DNA ladder kit was obtained from Roche Diagnostics (Tokyo, Japan). Cell Counting Kit-8 was obtained from Dojindo Laboratories (Kumamoto, Japan).
Cell culture
MIN6 cells were cultured in 60 ml flasks, 96-well plates, or on glass coverslips in DMEM (high-glucose; 4.5 g/l) with 15% FBS, 75 μg/ml penicillin, and 50 μg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. At 80% confluence, the cells were divided into four groups: CoQ10, Z-VAD, STS, and control. The CoQ10 group was treated with 30 μM CoQ10 for 4 h before induction of apoptosis. Z-VAD-FMK is a pan-caspase inhibitor and anti-apoptotic agent. The Z-VAD group was treated with 30 μM Z-VAD-FMK for 1 h before induction of apoptosis. STS and control groups were not pre-treated. Except for the control group, each group was treated with 0.5 μM STS for the specified periods to induce apoptosis.
WST-8 assay
Cell viability was compared among the four groups after apoptotic stimulation using Cell Counting Kit-8 that determines cell viability through reduction of s water-soluble tetrazolium salt, WST-8. WST-8 is reduced by dehydrogenases in cells to produce a yellow formazan dye. The amount of formazan dye generated by the activities of dehydrogenases in cells is directly proportional to the number of living cells. Except for the control group, MIN6 cells were treated in 96-well plates (100 μl medium per well) with 0.5 μM STS for 16 h. Then, 10 μl Cell Counting Kit-8 solution was added to each well, followed by incubation for 2 h. The number of living cells was determined by absorbance at 450 nm using fluorescence microplate reader. Several experiments were performed, and average values were calculated.
Annexin-V staining
At the early stage of apoptosis, cells lose their phospholipid membrane asymmetry and expose phosphatidylserine (PS) at the cell surface. This process can be monitored using annexin-V, a Ca2+-dependent, phospholipid-binding protein with high affinity for PS, which is useful for identifying apoptotic cells with exposed PS. For annexin-V staining, MIN6 cells were cultured on glass coverslips. After 6 h of treatment with STS, cells were washed with PBS and then stained with green fluorescent protein-labeled annexin-V for 5 min while protected from light, according to the manufacturer’s protocol. The cells were then washed with PBS and treated with methanol for 10 min. The methanol was removed, and propidium iodide (PI) was added, a red fluorescent nuclear counterstain. Samples were observed under a fluorescence microscope. Methanol-treated samples (all cells) were stained with PI (red), whereas early apoptotic cells were stained with annexin-V (green) and PI. We counted respectively 140, 113, 208 and 235 cells in Control, STS, CoQ10 and Z-VAD group. Thus, we were able to calculate the ratio of early apoptotic cells.
SDS-polyacrylamide gel electrophoresis (PAGE) and western blot analysis
To detect activation of caspase-3 and cytochrome c release from mitochondria after treatment with STS, cells were washed and then lysed in cell lysis buffer containing a protease inhibitor cocktail. The cell lysates (10 µg protein per sample) were then separated by 12.5% SDS-PAGE and electrotransferred onto a PVDF transfer membrane. Membranes were blocked with Tris-buffered saline containing with 0.05% Tween 20 and 5% nonfat dried milk and then incubated with primary antibodies for 1 h at room temperature. Primary antibodies were against caspase-3 (1:1000 dilution), cytochrome c (1:200 dilution), and β-actin (1:1000 dilution). The membrane was then incubated with secondary antibodies for appropriate times (30–60 min). Specific proteins were visualized by the enhanced chemiluminescence method using western blotting detection reagent.
DNA degradation analysis
After 14 h of treatment with STS, isolation of DNA from cells of each group was performed according to a standard procedure using a commercially available apoptotic DNA ladder kit. DNA fragmentation analysis was performed using a 1.5% agarose gel.
Statistical analysis
Data are expressed as the mean ± standard deviation. Comparisons of individual treatments in WST-8 assays were conducted using the Games-Howell post hoc test following one-way analysis of variance. Comparisons of individual treatments in annexin-V analysis were conducted using the Chi square test. P < 0.05 was considered as statistically significant.