I-2 knockdown phenotypes are prevented by cell cycle arrest at S phase. ARPE-19 cells were transfected with siRNA for I-2 or luciferase as control, and after 24 h were treated with 1.5 mM hydroxyurea in the presence of the siRNA for an additional 48 h. Cells were fixed for flow cytometry analysis, or for γ-tubulin staining or for Western blot. (A) DNA content profiles of cells were determined by flow cytometry, with DNA content measured by propidium iodide staining. (B) Cells transfected with siRNA in the presence of hydroxyurea were collected and immunoblotted for I-2, with actin as loading control. (C) After transfection with I-2 siRNA or control siRNA for 72 h, the percentage of cells (of 100 total) with abnormal centrosomes was counted. Cultures with or without hydroxyurea treatment were compared. Data are plotted as mean ± SD from three independent experiments.