CLASP2 is required for the formation of robust K-fibers that ultimately satisfy the SAC. (A) Metaphase-arrested U2OS parental cells and mRFP-CLASP2γ–expressing cell lines after CLASP RNAi were fixed and stained after a short cold shock. Ndc80 RNAi was used as a positive control. The respective mean and standard deviation (SDV) of the K-fiber signal intensity are indicated for all experimental conditions. Scale bar is 10 µm. (B) Graphical representation of the normalized K-fiber signal quantification. The corresponding signal of α-tubulin in the close vicinity of each KT was quantified and background subtracted (highlighted, black circle). (C) Quantification of K-fiber signal intensity. Each signal was normalized to the mean signal of Scramble siRNA in parental cells (bars represent mean and SD. Parental scramble = 389 KTs; siNdc80 = 407 KTs; Parental with CLASPs RNAi = 396 KTs; WT = 390 KTs; 2ea-3eeaa = 395 KTs; IP12 = 408 KTs; 2ea-IP12 = 415 KTs; IP12-3eeaa = 424 KTs; ΔC = 418KTs; ΔC-Gcn4 = 421 KTs; ΔC-Spc25 = 408 KTs; C-term = 427 KTs. n = 21 cells per condition, pool of three independent experiments. ****, P < 0.0001; two-tailed Welch’s t test. (D) Metaphase-arrested U2OS parental cells and mRFP-CLASP2γ–expressing cell lines after CLASPs RNAi were fixed and stained in order to quantify the percentage of cells with Mad1-positive KTs. The insets in the top panel represent higher magnification of selected regions. Scale bar is 10 µm. (E) Quantification of the percentage of cells with Mad1-positive KTs for each cell line. Bars represent mean and SD. n = 300 cells for each cell line from a pool of three independent experiments, with the exception of siNdc80 (n = 232). *, P < 0.05; two-tailed Welch’s t test.