Disrupting the H3pT3–Survivin interaction does not delay metaphase Knl1 dephosphorylation and SAC silencing in cells lacking H2ApT120-bound Sgo1. (A–D) DMSO or 5-ITu was added to HeLa or the Sgo1-K492A cells 9 h after release from STB. Cells were then fixed 12 h after release for immunostaining. Example images are shown (A and C). The relative intensities of Knl1-pRVSF (B) and Knl1-pMELT (D), representing the normalized ratios of Knl1-pRVSF/ACA and Knl1-pMELT/ACA, were determined in 60 cells from three independent experiments (20 cells per experiment). (E and F) DMSO or 0.6 µM 5-ITu was added to HeLa or the Sgo1-K492A cells stably expressing H2B-GFP at 7 h after release from STB, and then mitosis progression was analyzed in ≥67 cells (E). Selected frames of the videos are shown (F). (G) DMSO or 0.3 µM 5-ITu was added to HeLa or the Sgo1-K492A cells, and then mitosis progression was analyzed as in E. The means and ranges of mitotic duration were determined in >74 cells for each condition from two independent experiments. Means and SDs are shown (B, D, E, and G; unpaired t test). Time stated in h:min. N.S., not significant. Scale bars, 10 µm. See also Fig. S5.