TEM4 depletion leads to mitotic delay, prolonged mitosis, and cell death. (A) Timing of mitotic entry after G1 release in the indicated cell lines. Individual data points are shown from three independent experiments. (B) Timing of mitotic transitions from the cells in A; NEBD: nuclear envelope breakdown, MET: metaphase, ANA: anaphase. For (A and B), a one-way ANOVA test followed by Kruskal–Wallis for significance was performed together with Dunn’s multiple comparisons test. (C) Quantification of the cell fate phenotypes from the live cell imaging experiment in A. Statistical differences were measured by two-way RM-ANOVA and by Dunnett’s multiple comparisons test. Both cell death after NEBD (P < 0.0001) and cell death after multiple metaphase alignments (P = 0.0006) are significantly different between control and TEM4-depleted cells. (D) Cumulative mitotic index in nocodazole-treated HeLa -TREx cells expressing TEM4 shRNA after 72 h of induction as determined from live cell imaging. The graph displays the mean ± SD. The data was accumulated from three independent experiments; N ≥ 20 cells per condition per experiment. (E) Control, TEM4-21, and TEM4-22 cells were induced for 72 h, and 0.4 μΜ RO-3306 was added for the final 16 h, before release into fresh media for 30 min. Cells were fixed and stained with CREST serum (red), Hoechst (blue), and either anti-MPS1 or anti-BUBR1 (green) as shown. Scale bar = 5 μm. (F) Quantification of the average relative intensity of MPS1 and BUBR1 at kinetochores from E. Graph shows mean ± SEM; n = 3 independent experiments, N ≥ 20 cells/condition/experiment. A one-way ANOVA - Kruskal–Wallis test for significance was performed and the P values were shown from Dunn’s multiple comparisons.