Phenotype of cells after Survivin shutoff. (A) Distribution of Survivin (red), α-tubulin (green), and DNA (blue) in Survivin^ON (A and A′) and Survivin^OFF (A″ and A‴) mitotic cells. Bar, 5 μm. (B) Phase-contrast images of Survivin^ON and Survivin^OFF cultures (the latter after 60-h growth in doxycycline). Bar, 10 μm. (C) Cells from both survivin knockouts KO1 and KO2 die after exposure to doxycycline. (D) Annexin V–positive (apoptotic) cells appear 36 h after shutoff of survivin transcription in KO1. (E) INCENP (red) is diffuse, but kinetochores (CENP-H–GFP, green) appear normal in Survivin^OFF cells (bottom). Top, Survivin^ON control. Bar, 5 μm. (F) INCENP and H3S10ph levels decrease shortly after Survivin levels drop after addition of doxycycline to KO1 cells. Loading control, anti–α-tubulin. WT, wild type. (G) Micrograph showing decreased H3S10ph staining (green) in Survivin^OFF cells (bottom). Top, Survivin^ON control. Bar, 5 μm.