Chmp4c-depletion reduces cold-stable microtubule polymers. (A–D) Cells transfected with negative siRNA (control), Chmp4c siRNA (siChmp4c), Chmp4a siRNA (siChmp4a), or Chmp4b siRNA (siChmp4b) were treated with ice-cold medium for the indicated times. (E) Centromere length in metaphase-like spindles. Insets show magnified centromeres, and values indicate the centromere length. Bars: (inset) 0.5 µm. (F) Quantification of centromere length from cells as in E or incubated with nocodazole (nocod) for 4 h. Control: n = 291 centromeres; 20 cells; siChmp4c: n = 697 centromeres, 26 cells; nocodazole: n = 200 centromeres, 20 cells from three independent experiments. (G) Cells expressing GFP, WT or S210A mutant Chmp4c:GFP resistant to degradation by siChmp4c-2, or GFP:Vps4-K173Q were treated with ice-cold medium for 15 min. Mean tubulin intensity is shown, and values in the controls were set to one. n = 90 cells (B, D, and G) from three independent experiments. Error bars show the SD. ***, P < 0.001 compared with the control. Statistically significant differences were determined by ANOVA and Student’s t test. Bars, 5 µm.