Analysis of chromosome congression in GTSE1^5xLID cells and quantification of Aurora B kinase abundance and activity after GTSE1 depletion. (A) Immunoblot on asynchronous cell lysates of GTSE1^5xLID cells expressing a mCherry-β-tubulin BAC transgene and transfected with control (Ctrl), GTSE1, MCAK, or GTSE1 + MCAK siRNA. Immunoblots are with anti-GTSE1, anti-α-tubulin, or anti-MCAK. (B) Time from NEB to anaphase onset in cells treated as in A. Cells showing a bipolar spindle and misaligned chromosomes are indicated in red (n ≥ 173 cells over n = 2 experiments pooled for analysis and representation). Letters on the plot indicate differences: conditions with the same letter are not statistically different (statistics from Wilcoxon test adjusted for false discovery rate, P < 0.05). (C) Table presenting the average percentage of cells showing the indicated phenotypes in cells from B (the average is presented ± SD). n.d., not determined. (D) Immunoblot on mitotic lysates of U2OS cells transfected with control, GTSE1(B1), GTSE1(B3), or GTSE1(T1) siRNA. Cells were synchronized in mitosis with Nocodazole. Phosphorylation of H3 Serine 10 (H3pS10) is used as a marker of Aurora B activity. Immunoblots are with anti-GTSE1, anti-Aurora B, anti-H3pS10, or anti-α-tubulin antibodies. (E) Quantification of GTSE1 and Aurora kinase B levels in immunoblots of U2OS cells transfected with control or GTSE1 siRNA. Levels were first normalized to α-tubulin level and then to control siRNA. Cells were synchronized in mitosis with Nocodazole. The mean ± SD is presented, n = 2. (F) Immunofluorescence images of metaphase-like U2OS cells transfected with control, GTSE1(B1), GTSE1(B3), or GTSE1(T1) siRNA. DNA and H3pS10 staining are shown. Scale bar: 10 µm. (G) Quantification of the H3pS10 total fluorescence intensity in cells from F. The total H3pS10 fluorescence intensity per cell was measured from 3D reconstructions and corrected for background. Within each experiment, the average total H3pS10 fluorescence intensity per condition was calculated and normalized to the corresponding control siRNA. The mean of these normalized values (± SD) is presented (n ≥ 37 cells per condition over n = 2 experiments). (H) Immunofluorescence images of GTSE1^5xLID cells transfected with GTSE1 siRNA to deplete the endogenous GTSE1 and showing a misaligned chromosome outside (upper lane) or within the spindle (lower lane). Tubulin, CREST, and MAD1 staining are shown. 4× magnification of the misaligned chromosomes. Positions of the magnified area are indicated. Numeric data is shown in Table S1.