Induction of G1 cell cycle arrest but not apoptotic cell death in MCF7 cells by the P2Et fraction. (A) MCF7 cells treated with the P2Et fraction for 6, 12 or 24 h, stained with JC-1 and analyzed by flow cytometry. Ethanol was used as negative and valinomycin as positive controls (*** p < 0.001). (B) Caspase 3 activity was measured using the ELISA assay after cells had been treated with the P2Et fraction or doxorubicin (positive control) for 48 h (** p < 0.005). (C) PS externalization was estimated in MCF7 cells using flow cytometry after treatment with the P2Et fraction, doxorubicin (positive control) or ethanol (negative control) for 48 h. (D) Cell cycle analysis was performed on MCF7 cells treated with ethanol (negative control), the P2Et fraction (7.80 μg/ml (A)), doxorubicin (0.004 μg/ml (B) or 0.002 μg/ml (C)), the P2Et fraction (A) + doxorubicin (B or C) and vincristine (0.01 μg/ml, positive control) for 24 h. The cells were permeabilized, stained with propidium iodide (PI), acquired on a FACSAria I and analyzed with FLowJo software. Histograms represent relative cell DNA content measured in three independent experiments.