CENP-I sumoylation and RNF4-dependent degradation after SENP6 depletion. (A) HeLa cells were treated with control or SENP6 siRNA for 36 h, followed by a 13-h thymidine block. Where indicated, RNF4 siRNA was transfected at 12 h after control or SENP6 siRNAs. Where indicated, MG132 was added during the final hour before cells were harvested. Cell lysates were subjected to immunoprecipitation using antibodies against CENP-I. The precipitates were subject to blotting with antibodies against CENP-I (left) or SUMO-2/3 (right). The arrow indicates unmodified CENP-I, and the asterisk indicates modified forms of CENP-I. Undepleted (+) or depleted (−) levels of SENP6 and RNF4 are indicated, along with a cartoon representation of SUMO (greens rounds) and ubiquitin (red rounds) modifications of CENP-I (light blue ovals) predicted after each treatment. (B) HeLa cells were treated as in A, and immunoblot analysis was conducted on whole cell extracts from each sample. Primary antibodies used for blotting are indicated to the right of each panel. (C) HeLa cells were treated with control or SENP6 siRNA and synchronized by DTB for 48 h. Where indicated, RNF4 siRNA was transfected at 12 h after control or SENP6 siRNAs. Cells were fixed and stained for CENP-I and centromeres (CREST) as indicated. Insets show the DNA. (D) Cells treated as in C 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 95% confidence interval (n = 300). Bar, 5 µm.