SENP6 depletion causes defects in chromosome congression and spindle assembly. (A) HeLa cells were treated with control or SENP6 siRNA and synchronized by DTB. Cells were fixed and stained for γ-tubulin and DNA. The asterisks indicate misaligned chromosomes. Distance between the poles (a) and the metaphase plate width (b) were measured and tabulated (mean ± SD; n = 40; P < 0.0001). Similarly treated cells were stained for α-tubulin and centromere (CREST; not depicted), and the distance between the sister kinetochore pairs on the metaphase plate were measured and tabulated (mean ± SD; n = 40; P < 0.0001). (B) HeLa cells were treated with anti-SENP6 (−) or control (+) siRNAs and DTB. Cells were transfected with the rescue constructs during the second thymidine block and harvested 48 h after siRNA treatment. The cells were lysed in sample buffer and immunoblotted using anti-SENP6 antibodies. (C) Cells treated as in B were fixed and stained with Hoechst 33342. The percentage of cells with one or more misaligned chromosomes was determined for each sample. The graph represents three independent experiments. Error bars represent the 95% confidence interval (n = 300). Bar, 5 µm.