Additional file 17: of Osteoblasts are “educated” by crosstalk with metastatic breast cancer cells in the bone tumor microenvironment

Figure S17. Altered cell growth of MCF-7 cells treated with EO CM is due to altered cell cycle. MCF-7 cells were plated at 1 × 105 cells/cm2 in 35 × 10 mm dishes. Twenty-four hours later, growth media were removed and cancer cells treated with 1 ml breast cancer growth media plus either a) vehicle media (EMEM, control) or b) EO-conditioned media. Breast cancer cells were fixed for at least 2 h with 95% cold ethanol beginning on day 1 after plating, and continuing every day for 5 days. For propidium iodide staining, ethanol was decanted and fixed cells washed once with PBS. Cells were resuspended in a solution of 50 ng/ml propidium iodide, 100 ng/ml RNAse A, and PBS per 1 × 106 cells and incubated for 30 min in the dark at room temperature. Stained cells were analyzed for propidium iodide staining using a BD LSRII flow cytometer at excitation 535 nm and emission at 617 nm. A minimum of 10,000 events were counted per sample. Cell cycle phase was analyzed using BDFACS Diva software and FlowJo software. Three biological replicates per time point were counted per condition. Shown are representative images. (TIF 2263 kb)