Figure 3.
TEM4 depletion leads to errors in spindle formation and orientation and chromosome segregation defects. (A) Quantification of the most common spindle phenotypes from Fig. 2 A. A total of ∼200 cells were analyzed from 3 independent experiments. P values shown are for the pairwise comparisons between the control and TEM4-depleted cells undergoing normal division from a one-way ANOVA test. (B) Spindle angle calculations from control, TEM4-21, and TEM4-22 cells. N ≥ 20 cells/condition, n = 3 independent experiments. (C) ShRNA expression was induced for 72 h using 0.5 μg/ml doxycycline and cells were treated for 3 h with 3 μM of nocodazole. After nocodazole washout cells were incubated with fresh media for 2 h. Fixed cells were stained with CREST serum (blue), anti-α-tubulin (green), and anti-γ-tubulin (red). Scale bar = 5 μm. The graph shows the quantification of α-tubulin intensity 2 h after release, mean ± SEM, n = 3 independent experiments, N ≥ 20 cells/condition. P values were determined from one-way ANOVA-Kruskal–Wallis followed by Dunn’s multiple comparisons tests. (D) Quantification of the spindle phenotypes observed in C. Statistical differences were measured by a two-way ANOVA, followed by Tukey’s multiple comparisons tests. (E) Quantification of chromosome misalignment in parental, TEM4-21, and TEM4-22 cells after shRNA induction. Cells were treated for 12 h with 5 μM STLC and released into fresh media containing MG132 for 4 h, prior to fixation and staining. The means ± SEM of three independent experiments, with ≥25 cells/condition are shown. P values were determined by a Brown-Forsythe ANOVA test followed by Dunnett’s T3 multiple comparisons test. (F) Quantification of lagging chromosomes in anaphase. Cells were treated for 12 h with 0.4 μΜ RO-3306 and released into fresh media for 1.5 h for control cells and 2.5 h for TEM4 shRNA inducible cell lines to enrich for cells in anaphase. Data shows means ± SEM, n = 3 independent experiments, N ≥ 25 cells/condition. P values were determined by a Kruskal–Wallis ANOVA test followed by Dunn’s T3 multiple comparisons test. Refer to the image caption for details.

TEM4 depletion leads to errors in spindle formation and orientation and chromosome segregation defects. (A) Quantification of the most common spindle phenotypes from Fig. 2 A. A total of ∼200 cells were analyzed from 3 independent experiments. P values shown are for the pairwise comparisons between the control and TEM4-depleted cells undergoing normal division from a one-way ANOVA test. (B) Spindle angle calculations from control, TEM4-21, and TEM4-22 cells. N ≥ 20 cells/condition, n = 3 independent experiments. (C) ShRNA expression was induced for 72 h using 0.5 μg/ml doxycycline and cells were treated for 3 h with 3 μM of nocodazole. After nocodazole washout cells were incubated with fresh media for 2 h. Fixed cells were stained with CREST serum (blue), anti-α-tubulin (green), and anti-γ-tubulin (red). Scale bar = 5 μm. The graph shows the quantification of α-tubulin intensity 2 h after release, mean ± SEM, n = 3 independent experiments, N ≥ 20 cells/condition. P values were determined from one-way ANOVA-Kruskal–Wallis followed by Dunn’s multiple comparisons tests. (D) Quantification of the spindle phenotypes observed in C. Statistical differences were measured by a two-way ANOVA, followed by Tukey’s multiple comparisons tests. (E) Quantification of chromosome misalignment in parental, TEM4-21, and TEM4-22 cells after shRNA induction. Cells were treated for 12 h with 5 μM STLC and released into fresh media containing MG132 for 4 h, prior to fixation and staining. The means ± SEM of three independent experiments, with ≥25 cells/condition are shown. P values were determined by a Brown-Forsythe ANOVA test followed by Dunnett’s T3 multiple comparisons test. (F) Quantification of lagging chromosomes in anaphase. Cells were treated for 12 h with 0.4 μΜ RO-3306 and released into fresh media for 1.5 h for control cells and 2.5 h for TEM4 shRNA inducible cell lines to enrich for cells in anaphase. Data shows means ± SEM, n = 3 independent experiments, N ≥ 25 cells/condition. P values were determined by a Kruskal–Wallis ANOVA test followed by Dunn’s T3 multiple comparisons test.

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