Cellquest analysis software v 3

The cell cycle distributions in different phases after exposure to Rosmanol were analyzed by flow cytometry. In brief, MCF-7 and MDA-MB 231 cells (5×10⁵ cells/well) were seeded into 6-well plates and exposed to 15, 30 and 60 µM Rosmanol for 48 h at 37°C. Further, the cells were harvested, washed twice with PBS, and fixed with 70% ethanol for 2 h at 4°C. After that, cells were centrifuged at 3,000 × g for 5 min at 4°C and washed with PBS, resuspended in 500 µl of buffer containing 0.5 mg/ml RNase and 25 mg/ml propidium iodide (PI, Beyotime Institute of Biotechnology), and incubated in the dark at 37°C for 15 min. The distribution of the cell cycle was determined by using Cytomics FC 500 flow cytometer (Beckman Coulter Inc.), and the data were analyzed using Cellquest analysis software v.3.0 (BD Biosciences).

Apoptotic MCF-7 and MDA-MB 231 cells were investigated using the Annexin V/propidium iodide (PI) apoptosis detection kit (Beyotime Institute of Biotechnology) followed by flow cytometry, according to the manufacturer's protocol. MCF-7 and MDA-MB 231 cells (5×10³ cells/well) were cultured in 6-well plates and treated with different concentrations (15, 30 and 60 µM) of Rosmanol for 48 h at 37°C. Cells were harvested by trypsinization with no EDTA and washed twice with PBS and then stained with 5 µl Annexin V-FITC (Beyotime Institute of Biotechnology) and 10 µl PI in 500 µl binding buffer for 15 min at room temperature (RT) in the dark. Apoptotic cells were determined by using Cytomics FC 500 flow cytometer (Beckman Coulter Inc.) and the data were analysed using Cellquest analysis software v.3.0 (BD Biosciences).

The changes induced by Rosmanol in the mitochondrial membrane were determined using Rhodamine 123 staining (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, MCF-7 and MDA-MB 231 cells (5×10³ cells/well) were seeded in 6-well plates and then treated with or without NAC and incubated at 37°C for 1 h. The cells were treated with or without (15, 30 and 60 µM) Rosmanol for 48 h at 37°C and stained with Rhodamine 123 for 15 min at 37°C. Mitochondrial membrane potential was detected by Cytomics FC 500 flow cytometer (Beckman Coulter Inc.) and the data were analyzed using Cellquest analysis software v.3.0 (BD Biosciences).