Figure 5.
R8-9 is the key determinant of the TPX2 function in cells. (A) Immunofluorescence staining of α-tubulin and DNA (DAPI) in TPX2-mAID-mClover/mCherry-TPX2 variant double knock-in HeLa cells treated with Dox and IAA (right panels). TPX2-mAID-mClover (TPX2-mAC) knock-in HeLa cells treated without or with Dox and IAA were used as controls (left panels). (B–D) Quantification of the spindle length (B), the ratio of the average intensity of MTs in the vicinity of chromosomes to that in spindle pole regions (C; not available for TPX2-depleted cells or FL1-10A–expressing cells due to the severe phenotypes such as monopolar spindle and prometaphase-like spindle), and mitotic index (D) in indicated cells for experiments shown in A. N.A., not available. n = 99–129 spindles (B); n = 20–31 spindles (C); n = 3 experiments (1,812–3,166 cells were measured for each condition; D). (E) Immunofluorescence staining of γ-tubulin and DNA (DAPI) in TPX2 WT– or FL89A mutant–expressing cells was performed 10 min after incomplete removal of nocodazole with twice rinse in the drug-free medium. (F) Quantification of the number of chromosome-associated γ-tubulin foci in TPX2 WT– or FL89A mutant–expressing cells. n = 19 cells from two experiments. (G) Scheme of experimental setup for centrinone treatment. (H) Immunofluorescence staining of γ-tubulin and DNA (DAPI) in TPX2 WT– or FL89A mutant–expressing cells treated with centrinone. (I) Quantification of the percentage of bipolar spindles with one centrosome and monopolar spindles. n = 3 experiments (222–410 cells were measured for each condition). (J) Immunofluorescence staining of EB1 and DNA (DAPI) in TPX2 WT– or FL89A mutant–expressing cells. (K) Quantification of EB1 intensity in the vicinity of chromosomes in TPX2 WT– or FL89A mutant–expressing cells. n = 19–20 cells from two experiments. (L) Immunofluorescence staining of HURP and DNA (DAPI) in TPX2 WT– or FL89A mutant–expressing cells. (M) Quantification of HURP intensity in the vicinity of chromosomes in TPX2 WT– or FL89A mutant–expressing cells. n = 35–46 cells from two experiments. Scale bars, 2 μm. Data represent mean ± SD. ***P < 0.001, two-tailed t test (unpaired). Refer to the image caption for details.

R8-9 is the key determinant of the TPX2 function in cells. (A) Immunofluorescence staining of α-tubulin and DNA (DAPI) in TPX2-mAID-mClover/mCherry-TPX2 variant double knock-in HeLa cells treated with Dox and IAA (right panels). TPX2-mAID-mClover (TPX2-mAC) knock-in HeLa cells treated without or with Dox and IAA were used as controls (left panels). (B–D) Quantification of the spindle length (B), the ratio of the average intensity of MTs in the vicinity of chromosomes to that in spindle pole regions (C; not available for TPX2-depleted cells or FL1-10A–expressing cells due to the severe phenotypes such as monopolar spindle and prometaphase-like spindle), and mitotic index (D) in indicated cells for experiments shown in A. N.A., not available. n = 99–129 spindles (B); n = 20–31 spindles (C); n = 3 experiments (1,812–3,166 cells were measured for each condition; D). (E) Immunofluorescence staining of γ-tubulin and DNA (DAPI) in TPX2 WT– or FL89A mutant–expressing cells was performed 10 min after incomplete removal of nocodazole with twice rinse in the drug-free medium. (F) Quantification of the number of chromosome-associated γ-tubulin foci in TPX2 WT– or FL89A mutant–expressing cells. n = 19 cells from two experiments. (G) Scheme of experimental setup for centrinone treatment. (H) Immunofluorescence staining of γ-tubulin and DNA (DAPI) in TPX2 WT– or FL89A mutant–expressing cells treated with centrinone. (I) Quantification of the percentage of bipolar spindles with one centrosome and monopolar spindles. n = 3 experiments (222–410 cells were measured for each condition). (J) Immunofluorescence staining of EB1 and DNA (DAPI) in TPX2 WT– or FL89A mutant–expressing cells. (K) Quantification of EB1 intensity in the vicinity of chromosomes in TPX2 WT– or FL89A mutant–expressing cells. n = 19–20 cells from two experiments. (L) Immunofluorescence staining of HURP and DNA (DAPI) in TPX2 WT– or FL89A mutant–expressing cells. (M) Quantification of HURP intensity in the vicinity of chromosomes in TPX2 WT– or FL89A mutant–expressing cells. n = 35–46 cells from two experiments. Scale bars, 2 μm. Data represent mean ± SD. ***P < 0.001, two-tailed t test (unpaired).

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