Disrupting the H3pT3–Survivin interaction impedes the dephosphorylation of Knl1-RVSF motif at metaphase kinetochores in a Bub1-dependent manner. (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 Knl1-pRVSF, representing the normalized ratio of Knl1-pRVSF/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 9 h after release from STB. Cells were then fixed 12 h after release for immunostaining. Example images are shown (C). The relative intensity of Knl1-pRVSF/ACA 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 ratio of Knl1-pRVSF/ACA in prometaphase (F) and metaphase (G) cells was determined as in B. Means and SDs are shown (B, D, F, and G; unpaired t test). N.S., not significant. Scale bars, 10 µm. See also Fig. S4.