Figure 2.
NAP1 KO cells have mitotic defects similar to those lacking TBK1. (A and B) Representative Western blot and semiquantitative analysis of p-TBK1/TBK1 levels normalized to vinculin in asynchronous and mitotic cells from WT HeLa, NAP1 KO clone 10, and NAP1 KO clone 12. Nocodazole was used for synchronization. n = 3 independent experiments. Error bars ± SEM. (C and D) Representative Western blot and semiquantitative analysis of p-TBK1/TBK1 levels normalized to vinculin in asynchronous and synchronized mitotic cells from HeLa, NAP1 KO clone 12, and the stable NAP1 rescue line. RO-3306 was used for synchronization. n = 3 independent experiments. Error bars ± SEM. (E) Growth curve with normalized luminescence for WT HeLa, NAP1 KO clone 10, and NAP1 KO clone 12. Error bars indicate ± SEM. n = 2 experimental replicates. (F–I) Percentage of mitotic (F), multinucleated (G), binucleated (H), and abnormal mitotic (I) cells from an asynchronous population of WT HeLa, TBK1 KO, and NAP1 KO cells. Error bars indicate ± SEM; n = 3 independent experiments. For mitotic index, multinucleated and binucleated cell counts, and random fields of view were captured sampling ∼1,000 cells per biological replicate from each genotype. For abnormal mitotic cell counts, random fields of view were captured to sample ∼50 mitotic cells per biological replicate. n = 3 independent experiments from each genotype. (J) Representative confocal images of defects in NAP1 KO cells: insets (i) binucleated cell (top), multipolar metaphase (bottom); (ii) multipolar prometaphase (top), splayed/unfocused spindle (bottom); (iii) multipolar cytokinesis (top), monopolar prometaphase/metaphase (bottom). DAPI (blue) was used as a nuclear counterstain, α-tubulin for cytoskeleton staining (green), and CREST for kinetochore staining (red). Scale bar, 20 μm, insets, 5 μm. Percentages in the upper right corner of the insets display the percentage of that type of defect. (K) Pie charts representing the percentage of different types of mitotic defects found in WT HeLa, TBK1 KO, and NAP1 KO cells. At least 50 mitotic cells per biological replicate. n = 3 independent experiments from each genotype were analyzed. (L and M) Percentage of cytokinetic (L) and abnormal cytokinetic (M) cells from an asynchronous population of WT HeLa, TBK1 KO, and NAP1 KO. Error bars indicate ± SEM. n = 3 independent experiments. Random fields of view were captured sampling ∼1,000 cells per biological replicate from each genotype. (N) Representative confocal images of cytokinetic defects seen in TBK1 KO (i and ii) and NAP1 KO (iii and iv): (i) unequal cytokinesis, (ii) multipolar cytokinesis, (iii) multipolar cytokinesis, (iv) unequal cytokinesis. DAPI (blue) was used as a nuclear counterstain, α-tubulin for cytoskeleton staining (green), and CREST for kinetochore staining (red). Scale bar, 20 μm, insets, 5 μm. Percentages in the upper right corner of the insets display the percentage of that type of mitotic defect. (O) Pie chart representing the percentage of different types of cytokinetic defects found in HeLa, TBK1, and NAP1 KO cells. Random fields of view were captured sampling ∼30–40 cytokinetic cells per biological replicate from each genotype. n = 3 independent experiments. *P < 0.05 compared to HeLa. One dot = one independent experiment. One-way ANOVA was performed for all statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Source data are available for this figure: SourceData F2.

NAP1 KO cells have mitotic defects similar to those lacking TBK1. (A and B) Representative Western blot and semiquantitative analysis of p-TBK1/TBK1 levels normalized to vinculin in asynchronous and mitotic cells from WT HeLa, NAP1 KO clone 10, and NAP1 KO clone 12. Nocodazole was used for synchronization. n = 3 independent experiments. Error bars ± SEM. (C and D) Representative Western blot and semiquantitative analysis of p-TBK1/TBK1 levels normalized to vinculin in asynchronous and synchronized mitotic cells from HeLa, NAP1 KO clone 12, and the stable NAP1 rescue line. RO-3306 was used for synchronization. n = 3 independent experiments. Error bars ± SEM. (E) Growth curve with normalized luminescence for WT HeLa, NAP1 KO clone 10, and NAP1 KO clone 12. Error bars indicate ± SEM. n = 2 experimental replicates. (F–I) Percentage of mitotic (F), multinucleated (G), binucleated (H), and abnormal mitotic (I) cells from an asynchronous population of WT HeLa, TBK1 KO, and NAP1 KO cells. Error bars indicate ± SEM; n = 3 independent experiments. For mitotic index, multinucleated and binucleated cell counts, and random fields of view were captured sampling ∼1,000 cells per biological replicate from each genotype. For abnormal mitotic cell counts, random fields of view were captured to sample ∼50 mitotic cells per biological replicate. n = 3 independent experiments from each genotype. (J) Representative confocal images of defects in NAP1 KO cells: insets (i) binucleated cell (top), multipolar metaphase (bottom); (ii) multipolar prometaphase (top), splayed/unfocused spindle (bottom); (iii) multipolar cytokinesis (top), monopolar prometaphase/metaphase (bottom). DAPI (blue) was used as a nuclear counterstain, α-tubulin for cytoskeleton staining (green), and CREST for kinetochore staining (red). Scale bar, 20 μm, insets, 5 μm. Percentages in the upper right corner of the insets display the percentage of that type of defect. (K) Pie charts representing the percentage of different types of mitotic defects found in WT HeLa, TBK1 KO, and NAP1 KO cells. At least 50 mitotic cells per biological replicate. n = 3 independent experiments from each genotype were analyzed. (L and M) Percentage of cytokinetic (L) and abnormal cytokinetic (M) cells from an asynchronous population of WT HeLa, TBK1 KO, and NAP1 KO. Error bars indicate ± SEM. n = 3 independent experiments. Random fields of view were captured sampling ∼1,000 cells per biological replicate from each genotype. (N) Representative confocal images of cytokinetic defects seen in TBK1 KO (i and ii) and NAP1 KO (iii and iv): (i) unequal cytokinesis, (ii) multipolar cytokinesis, (iii) multipolar cytokinesis, (iv) unequal cytokinesis. DAPI (blue) was used as a nuclear counterstain, α-tubulin for cytoskeleton staining (green), and CREST for kinetochore staining (red). Scale bar, 20 μm, insets, 5 μm. Percentages in the upper right corner of the insets display the percentage of that type of mitotic defect. (O) Pie chart representing the percentage of different types of cytokinetic defects found in HeLa, TBK1, and NAP1 KO cells. Random fields of view were captured sampling ∼30–40 cytokinetic cells per biological replicate from each genotype. n = 3 independent experiments. *P < 0.05 compared to HeLa. One dot = one independent experiment. One-way ANOVA was performed for all statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Source data are available for this figure: SourceData F2.

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