Changes in kinetochore composition after SENP6 depletion. (A–E) HeLa cells were treated with control or SENP6 siRNA and synchronized by DTB for 48 h. Cells were fixed and stained for kinetochore proteins as indicated: CENP-O (A), Hec1 (B), Mis12 (C), CENP-F (D), and CENP-E (E). Cells were simultaneously stained for centromeres (CREST). The bottom right inset in each panel shows Hoechst 33342 staining. Other insets show magnification of the indicated areas. IFIs of the proteins were measured on kinetochores of prometaphase cells as well as on attached (aligned) or unattached (nonaligned) kinetochores of metaphase cells, as indicated. The graphs show mean ± 95% confidence interval (n = 100 for bipolarly attached sister kinetochores; n = 40 for unattached misaligned kinetochores; ***, P < 0.0001; and *, P < 0.05, as indicated; n. d. = measured value is not distinguishable from background signal). AU, arbitrary unit. Bars, 5 µm.