Figure 2. D004 treatment alters cell cycle arrest mechanisms.

NPA and DRO cells at 80% confluence are grown overnight and treated with 1 μM D004. After trypsinization, cells were washed with saline and fixed with 70% cold ethanol for 30 min at room temperature. Cells were collected by centrifugation and stained with propidium iodide (50 mg/mL in PBS for 30 min. Cells then were treated with DNAse free RNAse (1mg/mL) for 30 min and analyzed by flow cytometry. D004 treatment resulted in a decrease in G₀/G₁ arrested cells from 61% (DRO) and 55% (NPA) in controls to 35% and 32% respectively. Additionally, treatment resulted in increases in sub G₀ (6% controls to 13% treated), G2/M (19% controls to 31% treated) and S phase (13% controls to 20% treated) populations in DRO cells as well as in NPA cells (sub G₀: 1% controls to 15% treated), G₂/M (24% controls to 27% treated) and S phase (19% controls to 25% treated). Of note an increase in sub G₀ population in indicative of DNA fragmentation which likely represents apoptosis.