br Cell cycle analysis br Cells were seeded
2.6. Cell cycle analysis
Cells were seeded in six-well plates and treated with 4 μM DN3 at the indicated time, trypsinized, washed in PBS, and fixed in ice cold 70% ethanol overnight. The fixed Spectinomycin were washed twice with PBS, and then resuspended in mixture solution (0.5% Triton X-100, 0.5 mg/ ml RNase and 100 μg/ml PI in PBS), incubated at 37 °C for 30 min. DNA contents/cells were analyzed in Accuri C6 flow cytometer, with a total of 10,000 cells tested. The histograms of DNA distribution were mod-eled as a sum of G0/G1, S and G2/M phase, using FlowJo software.
gastric cancer cell lines and human
gastric epithelial mucosa cells GES-1.
cells were plated in 96-well culture
plates. 24 h later, the cells were
changed with fresh medium and
treated with DN3 at the indicated doses
was used as controls. Cell viability was
measured by MTT assay. Data are
and GES-1 cells are determined by
trypan blue dye exclusion assay. Data
are presented as means ± S.D. of tri-
gastric cancer cell lines.
As previously reported (Ma et al., 2016), MMP was measured using a potential-sensitive dye JC-1. Briefly, cells were exposed to DN3 alone at the indicated concentrations or DN3 in combination with DIDS or CsA for 24 h. At the end of the incubation period, the cells were col-lected and washed twice with cold PBS, and then incubated with medium containing 10 μg/ml JC-1 for 30 min at 37 °C. The MMP was measured under an excitation at 488 nm using a fluorescence micro-scopy, or analyzed by flow cytometry.
2.8. Determination of cytochrome c release from the mitochondria in intact cells
Cells were treated with DN3 at the indicated concentrations for various times. The mitochondria and cytosol fractions were prepared according to the manufacturer's instructions for the mitochondria iso-lation kit (Beyotime Institute of Biotechnology, Jiangsu, China). Briefly, cells were suspended in 200 μl ice-cold mitochondrial separation solu-tion for 10 min, and homogenized with a tight pestle on ice in a glass homogenizer (40 passes). The homogenate was subjected to centrifu-ging at 600g for 10 min at 4 °C to remove nuclei and unbroken cells. Then the supernatant was collected and centrifuged again at 12,000g for 30 min at 4 °C to obtain the cytosol (supernatant) and the mi-tochondria (pellet) fraction. Samples of the cytosol and the mitochon-dria were dissolved in lyses buffer were subjected to western blotting.
2.9. Detection of cytochrome c release from isolated mitochondria
Mitochondria (isolated from 1 × 107 cells per sample) were
incubated in mitochondria stock-solution contained 4 μM DN3 (Beyotime Institute of Biotechnology, Jiangsu, China) at 37 °C for the indicated time in the figure legends. Following the incubation, sample tubes were centrifuged at 12,000g for 30 min at 4 °C. The supernatants were collected, which contained the cytochrome c released from the isolated mitochondria, and the pellet consisted of the mitochondrial fraction. Cytochrome c release was determined by western blotting.
2.10. Measurement of mitochondrial swelling
Mitochondria were isolated from HGC-27 cells as described above. Mitochondria suspensions (equivalent to100 μg protein per well) were incubated in 96-well microtiter plates at 25 °C in assay buffer (Beyotime Institute of Biotechnology, Jiangsu, China). DN3 was added after 10 min of preincubation. As previously described (Flis et al., 2016), mitochondrial swelling leads to a decrease in absorbance monitored at 540 nm, which can be spectrophotometrically performed.
2.11. Western blotting analysis
After treatment under each experimental condition, cells were lysed in cell lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) supplemented with protease and phosphatase inhibitors cocktails (Roche). Protein content was determined with BCA reagent (Beyotime Biotechnology). Lysates were diluted with sample loading buffer (5 ×, Beyotime) at a 4:1 ratio and denatured at 100 °C for 3–5 min. Clarified protein lysates (containing equal amounts of protein, 20–100 μg) were electrophoretically separated by 10% SDS-polyacrylamide gel and proteins were transferred onto nitrocellulose membranes. The mem-branes were blocked with 5% nonfat milk for 2 h at room temperature,