The CHC/TACC3 complex recruits GTSE1 to the spindle to promote chromosome congression. (A) Western blots of U2OS clonal lines expressing RNAi-resistant GTSE1-GFP (GTSE1^WT) or GTSE1^5xLID-GFP (GTSE1^5xLID) from BAC transgenes. Cells were transfected with control (Ctrl) or GTSE1 siRNA, and lysates were probed for GTSE1 and α-tubulin. (B and C) Mass spectrometry analysis of GTSE1-interacting proteins following anti-GFP IP of mitotic lysates from GTSE1^WT or GTSE1^5xLIDcells, compared with U2OS cells. Curved lines represent significance threshold. (D) Immunofluorescence images of GTSE1^WT and GTSE1^5xLID cells transfected with GTSE1 siRNA. (E) Quantification of the GTSE1-GFP levels on spindles from of GTSE1^WT and GTSE1^5xLID cells depleted for endogenous GTSE1 (n = 3 experiments, n ≥ 26 half spindles from 13 cells per condition and per experiment, one experiment presented as a box and whisker plot, P value from Wilcoxon test). (F) Fraction of cells that have entered anaphase as a function of the time (minutes) after NEB. GTSE1^5xLID cells have a delayed time of anaphase onset. The mean (± standard error) time between NEB and anaphase onset is indicated in the insets. U2OS, GTSE1^WT, and GTSE1^5xLID cells were transfected with control or GTSE1 siRNA. (n > 223 cells per condition, n = 3 over four experiments; data were pooled for analysis and representation, P values from Wilcoxon test show differences to respective control siRNA). (G) Percentage of fixed metaphase-like cells showing chromosome misalignment. The mean percentage (± standard error) over n = 3 experiments is presented (P values from two-sided, unpaired t test, n ≥ 184 cells per condition). n.s., not significant. Scale bars: 5 µm. Numeric data is shown in Table S1.