GTSE1 stabilizes MTs in mitosis and promotes correct spindle orientation. (A) Immunofluorescence images of U2OS cells after control RNAi, GTSE1 RNAi, and stable knockout of GTSE1 stained for DNA (DAPI) and MTs (tubulin) showing fewer astral MTs after reduced GTSE1 levels. (B) Quantification of the percentage of cells lacking astral MTs from immunofluorescence analysis as shown in A. The left graph shows control and GTSE1 RNAi in U2OS cells, two stable U2OS cell clones expressing RNAi-resistant GTSE1 (GTSE1^WT(204) and GTSE1^WT(212)), and two stable U2OS cell clones expressing RNAi-resistant GTSE1 mutated to abolish interaction with EB1 (GTSE1^Sk(202) and GTSE1^Sk(208)). n > 150 cells; 3 experiments per condition. The right graph shows two independent U2OS GTSE1 knockout clones. P-values were obtained from a χ² test comparing against the U2OS control condition (marked with •). n > 100 cells. (C) Quantification of inner-spindle tubulin fluorescence intensity from fixed U2OS cells stained for α-tubulin after control or GTSE1 RNAi. n ≥ 19 per experiment per condition; 3 experiments. (D) Quantification of the mean length of astral MTs in three dimensions from immunofluorescence analysis as shown in A. 10 astral MTs were measured per cell. n = 10 cells per experiment per condition; 3 experiments. (E) Immunofluorescence images of U2OS cells after control RNAi, GTSE1 RNAi, and stable knockout of GTSE1 stained for EB1 showing fewer EB1 astral MT comets after GTSE1 RNAi. Graph shows quantification of astral MT length using the position of EB1 comets with respect to the centrosome. n > 5,800 astrals from 15 cells per condition; 1 experiment. P-values were obtained using a Kruskal-Wallis test followed by Conover-Iman test. (F) Quantification of the mean number of astral MTs per cell obtained by quantifying the number of EB1 comets in U2OS cells after control or GTSE1 RNAi and in GTSE1^KO(1) and GTSE1^KO(2) cells. n ≥ 13 cells per condition; 1 experiment. (E and F) Error bars represent standard deviation. P-values were obtained using an analysis of variance and a Tukey’s test. (G) Live-cell fluorescence images of metaphase U2OS cells expressing either BAC-expressed GTSE1-GFP (GTSE1^WT(212)) or endogenously tagged GTSE1-GFP. Both constructs localize to the spindle. (H) Analysis of spindle orientation. Images of mitotic cells viewed from the side and stained for DNA (blue), kinetochores (red), and centrioles (green) depict a cell with normal spindle alignment parallel to the substrate (top) and a cell with defective orientation (bottom). The angle of spindle tilt was calculated by determining the angle between the substrate and a line connecting both centrosomes, as depicted. Quantification of the percentage of metaphase cells with a spindle tilt angle >20° is shown. n > 140 cells; 3 experiments per condition. Bars, 5 µm. All error bars represent SEM unless otherwise specified. *, P ≤ 0.05; ***, P ≤ 0.001.