Centrosome amplification and mitotic defects due to I-2 knockdown in ARPE-19 cells. (A–D) Centrosome amplification was found in both interphase and mitotic cells. ARPE-19 cells were fixed 72 h after transfection with siRNA for I-2, or a control siRNA, and stained with antibody against γ-tubulin to identify centrosomes (orange). Centrosome amplification was evident in both interphase (B) and mitotic (D) I-2 knockdown cells, compared with corresponding control cells (A and C). Centrosome amplification was found in 18% of I-2 knockdown cells and 2% in control cells. (E–NH). ARPE-19 cells transfected with siRNA were fixed and stained with Hoechst 33342 plus antibody against Cep57 (green) to independently identify centrosomes. The Cep57 staining reveals centrosome amplification in both interphase (F) and mitotic (H) I-2 knockdown cells, compared with control (E and G). (I and J) Lagging chromosomes were detected in I-2 knockdown cells based on wide-field microscopic image of Hoechst 33342-stained DNA in anaphase cells (I) and a confocal image of an anaphase cell stained with ACA (J). In 100 I-2 knockdown anaphase cells, 7.5 ± 1.5% of the cells had lagging chromosomes, compared with 1.5 ± 1.5% in control cells. Bars, 25 μm (A, C, E and G) and 20 μm (I and J).