Disrupting the H3pT3–Survivin interaction hampers the removal of SAC proteins from metaphase kinetochores. (A and B) The indicated Survivin stable cell lines were released from STB for 12 h and then immunostained. Example images are shown (A). The relative intensity of Bub1, representing the normalized ratio of Bub1/ACA, was determined in 60 cells from three independent experiments (20 cells per experiment; B). (C and D) DMSO or 5-ITu was added to HeLa cells 7 h after release from STB. Cells were then fixed 12 h after release for immunostaining. Example images are shown (C). The relative intensity of Bub3 was determined as in B (D). (E–G) DMSO or BAY 1816032 was added to the indicated Survivin stable cell lines 9 h after release from STB. Cells were then fixed 12 h after release for immunostaining. Example images are shown (E). The relative intensity ratios of Bub3 (F) and Bub1 (G) were determined as in B. (H–J) DMSO or BAY 1816032 was added to the indicated Survivin stable cell lines 9 h after release from STB. Cells were then fixed 12 h after release for immunostaining. Example images are shown (H). The relative intensity ratios of Bub3 (I) and Bub1 (J) were determined as in B. Means and SDs are shown (B, D, F, G, I, and J; unpaired t test). N.S., not significant. Scale bars, 10 µm. See also Fig. S2.