Figure 2. Induction of G2/M cell cycle arrest and apoptosis by O. majorana extract in MDA-MB-231 cells.

(A) MDA-MB-231 cells (1.8×10⁶) seeded on 100 mm culture dish were exposed various concentrations of O. majorana extract or equal volume of vehicle (ethanol) as control for 24 h. Following treatment, cells were harvested, fixed, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry. Data represent the mean of three independent experiments. The percentage of cells in sub-G1 (apoptosis), G1, S and G2/M appears at the upper right of each graph. (B) Expression of cell cycle regulator in OME-treated MDA-MB-231. Western blot analysis of phospho(ser10)-H3, and cyclin B1 in MDA-MB231 cells exposed for 24 h to ethanol or indicated concentrations of OME. (C) Stimulation of caspase 3/7 activity in MDA-MB-231 cells after exposure to OME (0–600 µg/mL) for 24 h and 48 h, relative to a similar amount of viable ethanol-treated cells. The relative caspase 3/7 activity was normalized to the number of viable cells per well and is expressed as fold of induction compared to the control. (D) Concentration-dependent induction of PARP cleavage in OME-treated MDA-MB231 cells. Cells were treated with or without increasing concentrations of the extract and proteins were extracted as described in Materials and Methods. Western blot analysis was carried out using anti-PARP antibodies. (*p<0.05, **p<0.005 and ***p<0.0005).