Disrupting H3pT3–Survivin and H2ApT120–Sgo1 interactions additively reduces Aurora B–mediated phosphorylation of Hec1 at prometaphase kinetochores. (A–C) The indicated Survivin stable cell lines were released from STB for 12 h and then immunostained. Example images are shown (A). The relative intensities of Hec1 (B) and Hec1-pS44 (C), representing the normalized ratios of Hec1/ACA and Hec1-pS44/ACA, were determined in 60 cells from three independent experiments (20 cells per experiment). (D and E) DMSO or 5-ITu was added to HeLa cells 9 h after release from STB. Cells were then fixed 12 h after release for immunostaining. Example images are shown (D). The relative intensity of Hec1-pS44 was determined as in C (E). (F) MG132 was added to the indicated Survivin stable cell lines 10 h after release from STB. Cells were treated with cold medium 12 h after release and then fixed and immunostained. The collected images were deconvolved and analyzed. (G) DMSO or 5-ITu was added to HeLa cells 9 h after release from STB, and then MG132 was added 10 h after release. Cells were treated with cold medium 12 h after release and then fixed and immunostained. The collected images were deconvolved and analyzed. (H and I) The indicated Survivin stable cell lines were released from STB for 12 h and then immunostained. Example images are shown (H). The relative intensity of Hec1-pS44 was determined as in C (I). Means and SDs are shown (B, C, E, and I; unpaired t test). N.S., not significant. Scale bars, 10 µm.