SENP6 depletion results in loss of the CENP-H/I/K complex from kinetochores. (A and B) HeLa cells were treated with control or SENP6 siRNA and synchronized by DTB. Metaphase cells were fixed and stained with CREST sera and with antibodies against the indicated centromeric proteins, CENP-I (A) and CENP-H (B). The bottom right inset in each panel shows Hoechst 33342 staining. Other insets show magnification of the indicated areas. IFIs of the centromeric proteins were measured. The graphs show mean ± 95% confidence interval for metaphase cells as indicated (n = 100 for bipolarly attached sister kinetochores; n. d. = measured value is not distinguishable from background signal). AU, arbitrary unit. Bars, 5 µm.