Reagents and kit. VER was purchased from the National Institutes for Food and Drug Control. Antibodies against PTEN (22034-1-AP), PI3K (20584-1-AP), cyclinB1 (55004-1-AP), Cdc2 (catalog no. 10762-1-AP), Bcl-2 (12789-1-AP), survivin (10508-1-AP), PARP1 (13371-1-AP), BAX (50599-2-Ig), and GAPDH (60004-1-Ig) were all purchased from Proteintech Group, Inc. (Rosemont, IL, USA), while antibodies against phosphorylated AKT ser473 (#4060), and total AKT (#4691), cleaved caspase3 (#9664), cleaved caspase9 (#9509) were acquired from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibody against cleaved PARP1 (G215) was purchased from ImmunoWay Biotechnology Company (Plano, TX, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories Co., Ltd. (Kumamoto, Japan). Cell cycle and apoptosis detection kits were obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China).
Cell culture. BC cell lines MCF-7, MDA-MB-231, SKBR3, and immortalized breast epithelial cells MCF-10A were all obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco’s minimum essential medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum.
CCK-8 assay. CCK-8 assay was performed to investigate the anti-proliferation effects of VER in BC cells and MCF-10A cells. Briefly, MCF-7, MDA-MB-231, SKBR3 cells and immortalized epithelial cells MCF-10A were seeded in 96-well plates at 1 × 104 cells/well and treated with different concentration (0, 20, 40, 80, 120, 160, 200, or 240 µM) VER for 24 or 48 h. Followingly, 10µL of CCK-8 was added and incubated for 2 h at 37°C. Finally, the optical density at 450 nm (OD 450) was measured by microplate reader.
Colony formation assay. After treatment with 0, 50, 100, or 150 µM VER for 48 h, the BC cells were subcultured for 14 days. Then, viable colonies were fixed, and stained with 0.5% crystal violet.
Cell cycle analysis. The proportions of each cell cycle phase in BC cells were examined through flow cytometry analysis. Briefly, the MCF-7 and MDA-MB-231 cells were cultured overnight, and treated with 0, 50, 100, or 150 µM VER for 48 h. Afterward, viable BC cells were collected, fixed with 70% ethanol overnight, washed and treated with RNase A and propidium iodide for 30 min in the dark. The DNA contents were determined by a flow cytometer.
DAPI staining assay. MCF-7 and MDA-MB-231 cells were cultured on coverslips overnight. After treatment with 0, 50, 100, or 150 µM VER for 48 h, cells were fixed and stained with DAPI for 30 minutes in room temperature, washed with 1 × PBS, finally observed and photographed by a fluorescence microscopy.
Apoptosis analysis. The proportions of apoptotic BC cells were examined by an Annexin V-fluorescein apoptosis analysis. MCF-7 and MDA-MB-231 cells were treated with 0, 50, 100, or 150 µM VER for 48 h and then incubated with propidium iodide and Annexin V-fluorescein for 30 min in the dark. The apoptotic cells were detected by a flow cytometer.
Western blot analysis. Radioimmunoprecipitation assay buffer containing protease inhibitors was used to extract the total protein of MCF-7 and MDA-MB-231 cells after 0, 50, 100, or 150 µM VER treatment. BC cells protein samples were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes, blocked for 1 h at room temperature and incubated with primary Antibodies at 4°C overnight. Then, the membranes were washed twice and incubated with the secondary Antibodies at 37°C for 1 h. Target protein bands were detected by the ChemiDoc XRS + System.
Statistical analysis. GraphPad Prism software 6.0 (GraphPad Software Inc., La Jolla, CA, USA) was used to perform Statistical analyses. Data of three independent experiments were presented in bar graphs as the mean ± standard deviation (SD). The probability value < 0.05 was considered statistically significant.