Helenalin induces cell cycle arrest in G1 phase and modulates apoptosis in a dose-dependent manner. (A) 1.85 × 105 cells/ml were seeded onto 6-well plates and incubated for 24 h. Various concentrations of helenalin were added to the culture medium and incubated for an additional 24 h. Cells were then harvested and analyzed by flow cytometry. The cell cycle phase distribution was determined using CellQuest software. (B) Percent of cells in Sub-G1 after drug treatment representing three independent experiments. (C) Measurement of apoptotic cell death by staining with FITC-Annexin V and Propidium Iodide. Lower right hand quadrant for each dose treatment represents percent of apoptosis, while upper right hand quadrant represents early necrotic cells. (D) Percent of cells that are either apoptotic, necrotic or total cell death as measured by staining cells with FITC-Annexin V and Propidium Iodide. Percentages are the average of three independent biological replicates.