Reagents and antibodies
Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS) and antibiotics (penicillin/ streptomycin) were acquired from WelGENE Inc. (Gyungsan, Republic of Korea). H2O2, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2’7’-di-chlorodihydrofluorescein diacetate (DCF-DA), 4′,6′-diamidino-2-phenylindole (DAPI), paraformaldehyde, ethidium bromide (EtBr), RNase A, propidium iodide (PI) and Triton X-100 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Mitochondrial fractionation kit and annexin V-fluorescein isothiocyanate (FITC) and PI double staining kit were obtained from Active Motif, Inc. (Carlsbad, CA, USA) and Becton Dickinson (San Jose, CA, USA), respectively. Comet assay kit was purchased from Trevigen Inc. (Gaithersburg, MD, USA). Caspase enzyme-linked immunosorbent assay kits were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Polyvinylidene fluoride (PVDF) membranes and enhanced chemiluminescence (ECL) reagent were obtained from Millipore (Bedford, MA, USA) and Amersham Biosciences (Westborough, MA, USA), respectively. Primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Abcam, Inc. (Cambridge, UK), and Cell Signaling Technology (Danvers, MA, USA) (Supplementary File 1). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG antibodies were obtained from Santa Cruz Biotechnology, Inc. All other reagents were from Sigma-Aldrich Chemical Co. unless otherwise stated.
Corni Fructus was kindly obtained from the Gurye Sansuyu Farming Association Corporation (Gurye, Republic of Korea), and the voucher specimen (WECU-17-2) was deposited in the Herbarium, Department of Biochemistry, Dong-eui University College of Korean Medicine (Busan, Republic of Korea). CFE was extracted as described previously . Briefly, dried Corni Fructus was ground and extracted with boiled distilled water using a reflux system. The liquid extract was then filtered using filter paper (Whatman No. 4, Whatman International Ltd., Maidstone, UK) to remove debris, concentrated with a rotary evaporator (EYELA, Bohemia, NY, USA), freeze-dried, and stored at -80°C until use in experiments.
Cell culture and treatment
Immortalized mouse skeletal C2C12 myoblasts were obtained from the American Type Culture Collection (CRL-1772™, Manassas, VA, USA) and maintained in DMEM, which containing 10% FBS and 1% antibiotics, at 37°C in a humidified atmosphere containing 5% CO2. CFE and H2O2 were dissolved in an appropriate amount of Milli-Q Water and diluted in the medium to the desired concentrations before treatment.
The MTT assay was performed for analysis of cell viability, as previously described . Briefly, cells were treated with the indicated concentrations of CFE or H2O2 alone for 24 h or pretreated with CFE for 1 h and then treated with H2O2 for 24 h. After treatment, MTT solution (0.5 mg/ml) was added to each well followed by incubation at 37°C for 3 h. Subsequently, the supernatant was removed, and the formed formazan was dissolved with DMSO. The absorbance at 540 nm was then evaluated using a microplate reader (Beckman Coulter, Brea, CA, USA) at Core-Facility Center for Tissue Regeneration, Dong-eui University (Busan, Republic of Korea). Cell viability of each treatment group was expressed as a percentage of the absorbance of the control group.
Measurement of ROS content
DCF-DA staining was applied to measure the level of ROS produced in the cells. In brief, cells were pretreated with the indicated concentrations of CFE for 1 h prior to expose to 1 mM H2O2 for another 1 h. The cells were stained with 10 μM DCF-DA at 37°C for 20 min in the dark, then the levels of ROS production were assessed using a flow cytometer (Becton Dickinson) or observed under a fluorescence microscope (Carl Zeiss, Oberkochens, Germany), according to the method described previously .
The comet assay kit was used to evaluate the protective effect of CFE against H2O2-induced DNA damage, as previously described . Briefly, cells were treated with H2O2 for 24 h or pretreated with CFE for 1 h followed by treatment with H2O2 for an additional 24 h. Thereafter, cells were spread onto slide glasses pretreated with agarose, according to the manufacturer's instructions. After agarose was solidified, slides were submerged with a lysis solution supplied in the kit. Electrophoresis was then performed at 25°C for 20 min at 300 mA and 25 V. After electrophoresis, cells were stained with EtBr. The degree of comet formation was observed under a fluorescence microscope.
Western blot analysis
To analyze the expression of the target proteins by immunoblotting, cells cultured in the absence or presence of CFE for 1 h were treated with H2O2 for 24 h. Total protein was extracted, as previously mentioned  or cytosolic and mitochondrial proteins were isolated for cytochrome c expression analysis using a mitochondrial fraction kit, according to the manufacturer's instructions. After quantification of the isolated proteins, samples containing the equal amount of protein were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to PVDF membranes for 1 h at room temperature (RT). The membranes were then probed with primary antibodies overnight at 4°C. After washing the membranes with PBS-T (phosphate-buffered saline with Tween 20), membranes were immediately blotted with HRP-conjugated secondary antibodies for 1 h at RT. ECL was used to visualize the proteins of interest in accordance with the manufacturer's instructions.
Annexin V-FITC/PI staining
To quantitatively evaluate the degree of apoptosis, annexin V-FITC and PI double staining was performed. To this end, the cells treated as mentioned above were washed with PBS and stained using annexin V/PI solution for 20 min at RT, according to the manufacturer’s protocol. Cells were then subjected to flow cytometry to quantify annexin V-positive cells as apoptosis-induced cells followed published procedures .
DNA fragmentation assay
To confirm the induction of DNA fragmentation, DNA gel electrophoresis was performed after cells were treated with H2O2 in the absence or presence of CFE as described above. Briefly, the collected cells were washed with PBS and resuspended in lysis buffer, as previously described . The cells were incubated with 1 μg/ml RNase A for 2 h at 37°C, and then genomic DNA was extracted from the supernatant with phenol/chloroform/isoamyl alcohol. After precipitation with ethanol, the DNA was resolved by electrophoresis on 1.5% agarose gel at 70 V. The gel was stained with 0.1 µg/ml EtBr and then the DNA ladders were visualized with a UV transilluminator (Vilber, Collégien, France).
To evaluate whether apoptosis was induced by observing morphological changes in the nucleus, DAPI staining was performed. In brief, cells were treated as described above, washed with PBS, and then fixed with 4% paraformaldehyde solution for 10 min at RT, according to a published method . The cells were stained with 1 μg/mL DAPI solution for 10 min in the dark and washed again with PBS. Morphological changes of the nucleus were then observed under a fluorescence microscope.
Analysis of MMP
Flow cytometric analysis by JC-1 staining was applied to monitor mitochondrial integrity. as previously described . In brief, the collected cells were washed with PBS and stained with 10 μM JC-1 solution in the dark for 20 min at RT, according to the manufacturer's procedure. After removing the supernatant, cells were washed again with PBS and analyzed using a flow cytometer to measure MMP. The loss of MMP was expressed as a ratio of JC-1 aggregates, indicating the extent of mitochondrial depolarization to form JC-1 monomers.
Caspase activity assay
The activity of caspase-9 and caspase-3 was determined using the caspase activity assay kits according to the manufacturer’s instructions. Briefly, after lysing cells to be measured, the supernatant was reacted with a reaction buffer per the recommendation of the manufacturer. The optical density of the reaction mixture of each sample after the reaction was measured at 405 nm using a microplate reader and expressed as a relative value .
Data are presented as mean ± standard deviation (SD). All experiments were repeated three times. Statistical analysis was performed using Student's t‑test, with GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Differences with p < 0.05 were considered statistically significant.