Figure 3.
NAP1 loss in a near diploid cell line causes mitotic and cytokinetic defects. (A) Cartoon diagram of dTAG knock-in constructs designed to add FKBP12F36V to the N-terminus of NAP1. (B–D) Representative Western blot and semiquantitative analysis of p-TBK1/TBK1 (C) and NAP1 (D) levels normalized to vinculin in asynchronous and mitotic cells from FKBP12F36V-NAP1 DLD-1 cells. RO-3306 was used for synchronization prior to mitotic release. Cells were treated with dTAGV-1 for 3 h in asynchronous conditions and 3 h prior and during release in mitotic conditions. n = 3 independent experiments. Error bars ± SEM. (E and F) Representative Western blot and semiquantitative analysis of NAP1 levels normalized to vinculin in asynchronous cells with or without dTAGV-1 treatment for 20 h. n = 3 independent experiments. Error bars ± SEM. (G–K) Percentage of mitotic (G), binucleated (H), multinucleated (I), dead (J) and abnormal mitotic (K) cells from an asynchronous population of FKBP12F36V-NAP1 DLD-1 cells untreated or treated with 20 h of dTAGV-1. Error bars indicate ± SEM; n = 3 independent experiments. For mitotic index, multinucleated, and binucleated cell counts, random fields of view were captured sampling ∼800–900 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. (L) Representative confocal images of the defects seen in FKBP12F36V-NAP1 DLD-1 cells after 20 h of dTAGV-1 treatment: (i) monopolar spindle, (ii) multipolar spindle, (iii) chromatin bridge/acentric fragment during anaphase (top) and binucleated cell (bottom), (iv) unfocused spindle poles. 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. (M) Pie charts representing the percentage of different types of mitotic defects found in untreated and dTAGV-1 treated FKBP12F36V-NAP1 DLD-1 cells for 20 h. At least 50 mitotic cells per biological replicate. n = 3 independent experiments from each genotype were analyzed. (N and O) Percentage of cytokinetic (N) and abnormal cytokinetic (O) cells from untreated and dTAGv-1 treated FKBP12F36V-NAP1 cells for 20 h. Error bars indicate ± SEM. Random fields of view were captured sampling ∼800–900 cells per biological replicate. n = 3 independent experiments from each genotype. (P) Pie chart representing the percentage of different types of cytokinetic defects found in untreated and dTAGV-1 treated FKBP12F36V-NAP1 DLD-1 cells for 20 h. Random fields of view were captured sampling ∼30–40 cytokinetic cells per biological replicate. n = 3 independent experiments from each genotype. (Q) Representative confocal images of cytokinetic defects seen in FKBP12F36V-NAP1 DLD-1 cells after 20 h of dTAGV-1 treatment: (i and ii) unequal cytokinesis, (iii) multipolar 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. Percentages in the upper right corner of the insets display the percentage of that type of mitotic defect. (R) Representative time-lapse images of the FKBP12F36V-NAP1 DLD-1 cells from UT (left) and dTAGv-1 treated (right). The actual experimental time from the beginning of the time-lapse imaging is displayed at the top left corner. The quantified duration of each early mitotic stage is displayed at the bottom. Scale bar, 20 μM. H2B–mCherry was used as the nuclear marker. (S–V) Duration of (S) prophase, (T) prometaphase, (U) metaphase, and (V) mitotic entry until the onset of anaphase in UT and dTAGv-1 treated FKBP12F36V-NAP1 DLD-1 cells from time-lapse imaging. Cells were pretreated with either DMSO for untreated or dTAGv-1 treated group for 1 h prior to the imaging. The cells were followed up to 4–5 h. Random fields of view were imaged to capture at least 100 mitotic cells from three independent biological replicates for each group. Error bars indicate ± SEM. One dot = one cell. (W) Frequency distribution graph for different observed mitotic errors from the time-lapse imaging analysis sampled from ∼100 mitotic cells in UT and dTAGv-1 treated FKBP12F36V-NAP1 DLD-1 cells. Kolmogorov–Smirnov nonparametric test was used to analyze the differences between the frequency distribution. (X) Percentage of cells with a mitotic error in UT and dTAGv-1 treated FKBP12F36V-NAP1 DLD-1 cells from the time-lapse imaging. At least 100 cells from three independent biological replicates for each group were analyzed. Error bars indicate ± SEM. One dot = one experimental replicate for (C, D, F, G–K, N, O, and X). Student’s t test was performed for all statistical analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Source data are available for this figure: SourceData F3.

NAP1 loss in a near diploid cell line causes mitotic and cytokinetic defects. (A) Cartoon diagram of dTAG knock-in constructs designed to add FKBP12F36V to the N-terminus of NAP1. (B–D) Representative Western blot and semiquantitative analysis of p-TBK1/TBK1 (C) and NAP1 (D) levels normalized to vinculin in asynchronous and mitotic cells from FKBP12F36V-NAP1 DLD-1 cells. RO-3306 was used for synchronization prior to mitotic release. Cells were treated with dTAGV-1 for 3 h in asynchronous conditions and 3 h prior and during release in mitotic conditions. n = 3 independent experiments. Error bars ± SEM. (E and F) Representative Western blot and semiquantitative analysis of NAP1 levels normalized to vinculin in asynchronous cells with or without dTAGV-1 treatment for 20 h. n = 3 independent experiments. Error bars ± SEM. (G–K) Percentage of mitotic (G), binucleated (H), multinucleated (I), dead (J) and abnormal mitotic (K) cells from an asynchronous population of FKBP12F36V-NAP1 DLD-1 cells untreated or treated with 20 h of dTAGV-1. Error bars indicate ± SEM; n = 3 independent experiments. For mitotic index, multinucleated, and binucleated cell counts, random fields of view were captured sampling ∼800–900 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. (L) Representative confocal images of the defects seen in FKBP12F36V-NAP1 DLD-1 cells after 20 h of dTAGV-1 treatment: (i) monopolar spindle, (ii) multipolar spindle, (iii) chromatin bridge/acentric fragment during anaphase (top) and binucleated cell (bottom), (iv) unfocused spindle poles. 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. (M) Pie charts representing the percentage of different types of mitotic defects found in untreated and dTAGV-1 treated FKBP12F36V-NAP1 DLD-1 cells for 20 h. At least 50 mitotic cells per biological replicate. n = 3 independent experiments from each genotype were analyzed. (N and O) Percentage of cytokinetic (N) and abnormal cytokinetic (O) cells from untreated and dTAGv-1 treated FKBP12F36V-NAP1 cells for 20 h. Error bars indicate ± SEM. Random fields of view were captured sampling ∼800–900 cells per biological replicate. n = 3 independent experiments from each genotype. (P) Pie chart representing the percentage of different types of cytokinetic defects found in untreated and dTAGV-1 treated FKBP12F36V-NAP1 DLD-1 cells for 20 h. Random fields of view were captured sampling ∼30–40 cytokinetic cells per biological replicate. n = 3 independent experiments from each genotype. (Q) Representative confocal images of cytokinetic defects seen in FKBP12F36V-NAP1 DLD-1 cells after 20 h of dTAGV-1 treatment: (i and ii) unequal cytokinesis, (iii) multipolar 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. Percentages in the upper right corner of the insets display the percentage of that type of mitotic defect. (R) Representative time-lapse images of the FKBP12F36V-NAP1 DLD-1 cells from UT (left) and dTAGv-1 treated (right). The actual experimental time from the beginning of the time-lapse imaging is displayed at the top left corner. The quantified duration of each early mitotic stage is displayed at the bottom. Scale bar, 20 μM. H2B–mCherry was used as the nuclear marker. (S–V) Duration of (S) prophase, (T) prometaphase, (U) metaphase, and (V) mitotic entry until the onset of anaphase in UT and dTAGv-1 treated FKBP12F36V-NAP1 DLD-1 cells from time-lapse imaging. Cells were pretreated with either DMSO for untreated or dTAGv-1 treated group for 1 h prior to the imaging. The cells were followed up to 4–5 h. Random fields of view were imaged to capture at least 100 mitotic cells from three independent biological replicates for each group. Error bars indicate ± SEM. One dot = one cell. (W) Frequency distribution graph for different observed mitotic errors from the time-lapse imaging analysis sampled from ∼100 mitotic cells in UT and dTAGv-1 treated FKBP12F36V-NAP1 DLD-1 cells. Kolmogorov–Smirnov nonparametric test was used to analyze the differences between the frequency distribution. (X) Percentage of cells with a mitotic error in UT and dTAGv-1 treated FKBP12F36V-NAP1 DLD-1 cells from the time-lapse imaging. At least 100 cells from three independent biological replicates for each group were analyzed. Error bars indicate ± SEM. One dot = one experimental replicate for (C, D, F, G–K, N, O, and X). Student’s t test was performed for all statistical analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Source data are available for this figure: SourceData F3.

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