Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: The primary antibodies used in this study were, Guinea pig anti-human CENP-C (1:10,000), rabbit anti-Cas9 (1:2,000, Takara Bio), mouse anti-human CENP-A (1:1,000), rabbit anti-human DSN1 (1:5,000), rabbit anti-human KIF18A (1:2000) (Bethyl), rabbit anti-human KIF15 (1:2000) (Proteintech), rabbit anti-human NDE1 (1:2000) (Proteintech), rabbit anti-human NEDD1 (1:2000) (Proteintech), rabbit anti-human SPAG5 (1:2000) (Proteintech), rabbit anti-human KNTC1 (1:2000) (Proteintech), rabbit anti-human ASPM (1:2000) (Proteintech), rabbit anti-human TUBGCP6 (1:2000) (Novus biologicals), mouse anti-human NPM1 (1:2000) (Proteintech) and mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich).

Techniques: Knock-Out, Two Tailed Test, Staining, Marker

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Schematic representation of human wild-type CENP-C (CENP-C WT ) and CENP-C ΔM12BD mutant, which are expressed in RPE-1 cells. See also . (B) Immunoblot analysis to detect KIF18A in RPE-1 CENP-C WT or CENP-C ΔM12BD cells after treatment with control non-targeting siRNA (siNT) or KIF18A siRNA (siKIF18A). α-tubulin was probed as a loading control. (C) Cell viability assay of RPE-1 CENP-C WT or CENP-C ΔM12BD cells after treatment with siNT or siKIF18A (mean and SD, two-tailed Student’s t test; CENP-C WT cells with siNT: n = 6; CENP-C WT cells with siKIF18A: n = 6; CENP-C ΔM12BD cells with siNT: n = 6; CENP-C ΔM12BD cells with siKIF18A: n = 6; ** p < 0.01). (D) Schematic representation of human wild-type DSN1 (DSN1 WT ) and DSN1 2A (S100A/S109A) mutant, which are expressed in RPE-1 cells. See also . (E) mScarlet-DSN1 localization in RPE-1 DSN1 WT or DSN1 2A cells. mScarlet-DSN1 is shown in green. CENP-T was stained as a kinetochore marker (red), DNA was stained with DAPI (blue). Scale bar, 10 μm. Signal intensities of mScarlet-DSN1 at mitotic kinetochores were quantified (mean and SD, two-tailed Student’s t test; DSN1 WT cells: n = 10 cells; DSN1 2A cells: n = 10 cells; * p < 0.1). (F) Cell viability of RPE-1 DSN1 WT or DSN1 2A cells after treatment with siNT or siKIF18A (mean and SD, two-tailed Student’s t test; DSN1 WT cells with siNT: n = 6; DSN1 WT cells with siKIF18A: n = 6; DSN1 2A cells with siNT: n = 6; DSN1 2A cells with siKIF18A: n = 6; ** p < 0.01). (G) Representative time-lapse images of mitotic progression in RPE-1 CENP-C WT or CENP-C ΔM12BD cells after treatment with siNT or siKIF18A. DNA was visualized with GFP-H2A. Images were projected using maximum intensity projection. Time is relative to nuclear envelope breakdown (NEBD). Scale bar, 10 μm. The time-lapse images were analyzed to measure the mitotic timing from NEBD to anaphase onset (CENP-C WT cells with siNT: n = 99 cells; CENP-C WT cells with siKIF18A: n = 84 cells; CENP-C ΔM12BD cells with siNT: n = 86 cells; CENP-C ΔM12BD cells with siKIF18A: n = 95 cells). The red bars show the cells that were arrested in mitosis and did not exit mitosis by the end of the imaging. (H) Cell viability of HeLa cells in the presence of various concentrations of Reversine after treatment of siNT or siKIF18A (mean and SD; each sample size: n = 3). (I) Cell viability of RPE-1 CENP-C ΔM12BD cells in the presence of various concentrations of Reversine after treatment with siNT or siKIF18A (mean and SD; each sample size: n = 6).

Article Snippet: The primary antibodies used in this study were, Guinea pig anti-human CENP-C (1:10,000), rabbit anti-Cas9 (1:2,000, Takara Bio), mouse anti-human CENP-A (1:1,000), rabbit anti-human DSN1 (1:5,000), rabbit anti-human KIF18A (1:2000) (Bethyl), rabbit anti-human KIF15 (1:2000) (Proteintech), rabbit anti-human NDE1 (1:2000) (Proteintech), rabbit anti-human NEDD1 (1:2000) (Proteintech), rabbit anti-human SPAG5 (1:2000) (Proteintech), rabbit anti-human KNTC1 (1:2000) (Proteintech), rabbit anti-human ASPM (1:2000) (Proteintech), rabbit anti-human TUBGCP6 (1:2000) (Novus biologicals), mouse anti-human NPM1 (1:2000) (Proteintech) and mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich).

Techniques: Mutagenesis, Western Blot, Control, Viability Assay, Two Tailed Test, Staining, Marker, Imaging

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Schematic representation of wild-type KIF18A and its functional domains, including the motor domain, coiled-coil domain, and PP1 binding domain. RPE-1 cells expressing KIF18A lacking the motor domain (KIF18A Δ1-362 ) or PP1 binding domain (KIF18A ΔPP1 ) were generated. KIF18A is phosphorylated by CDK1 at S674 and S684. RPE-1 cells expressing a KIF18A mutant in which S674 and S684 were replaced with Ala (KIF18A 2A ) or KIF18A 2A lacking PP1 binding domain (KIF18A ΔPP1+2A ) were generated. See . (B and C) Immunoblot analysis to detect WT or mutant KIF18A protein in RPE-1 CENP-C WT (B) or CENP-C ΔM12BD (C) cells with siNT or siKIF18A treatment. α-tubulin was probed as a loading control. (D and E) Cell viability assay of RPE-1 CENP-C WT (D) or CENP-C ΔM12BD (E) cells expressing WT or various KIF18A mutants with siNT or siKIF18A treatment (mean and SD, two-tailed Student’s t test; each sample size: n = 6; * p < 0.1; ** p < 0.01). (F) Localization of KIF18A WT and its mutants (green) at plus-end of microtubules. RPE-1 CENP-C WT cells expressing WT or various KIF18A mutants were treated with siKIF18A for 2 days before fixation. KIF18A was stained with an antibody against human KIF18A. mScarlet-CENP-A was used as a kinetochore marker (red), and microtubules were stained with an anti-α-tubulin antibody (cyan). DNA was stained with DAPI (blue). Scale bar, 10 μm. (G) Cell viability assay of RPE-1 CENP-C WT or CENP-C ΔM12BD cells with UMK57 at various concentrations (mean and SD; each sample size: n = 6).

Article Snippet: The primary antibodies used in this study were, Guinea pig anti-human CENP-C (1:10,000), rabbit anti-Cas9 (1:2,000, Takara Bio), mouse anti-human CENP-A (1:1,000), rabbit anti-human DSN1 (1:5,000), rabbit anti-human KIF18A (1:2000) (Bethyl), rabbit anti-human KIF15 (1:2000) (Proteintech), rabbit anti-human NDE1 (1:2000) (Proteintech), rabbit anti-human NEDD1 (1:2000) (Proteintech), rabbit anti-human SPAG5 (1:2000) (Proteintech), rabbit anti-human KNTC1 (1:2000) (Proteintech), rabbit anti-human ASPM (1:2000) (Proteintech), rabbit anti-human TUBGCP6 (1:2000) (Novus biologicals), mouse anti-human NPM1 (1:2000) (Proteintech) and mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich).

Techniques: Functional Assay, Binding Assay, Expressing, Generated, Mutagenesis, Western Blot, Control, Viability Assay, Two Tailed Test, Staining, Marker

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of RPE-1, K562, U2OS, A549, TIG-3, HeLa, HT1080, OVCAR-3, HT29, and HCC1806 cells with or without KIF18A knockdown (mean and SD, two-tailed Student’s t test; each sample size: n = 6; ** p < 0.01). (B–D) CENP-E (B), CENP-C (C), and CENP-T (D) localization in RPE-1, K562, U2OS, A549, TIG-3, HeLa, HT1080, OVCAR-3, HT29, and HCC1806 cells. The cell lines were treated with 50 μM monastrol for 2 h to enrich prometaphase cells. Each cell line was cytospun together with monastrol-treated RPE-1 cells expressing mScarlet-CENP-A as an internal control for immunostaining. CENP-E (green) (B), CENP-C (green) (C), and CENP-T (green) (D) were stained with antibodies against each protein. DNA was stained with DAPI (blue). CENP-T or CENP-A was stained as a kinetochore marker (red). Scale bar, 10 μm. CENP-E, CENP-C, or CENP-T signal intensities at mitotic kinetochores were quantified and normalized with those of the internal control RPE-1 mScarlet-CENP-A cells in each sample (mean and SD, two-tailed Student’s t test, each cell line: n = 10 cells; n.s., non-significant; * p < 0.1; ** p < 0.01).

Article Snippet: The primary antibodies used in this study were, Guinea pig anti-human CENP-C (1:10,000), rabbit anti-Cas9 (1:2,000, Takara Bio), mouse anti-human CENP-A (1:1,000), rabbit anti-human DSN1 (1:5,000), rabbit anti-human KIF18A (1:2000) (Bethyl), rabbit anti-human KIF15 (1:2000) (Proteintech), rabbit anti-human NDE1 (1:2000) (Proteintech), rabbit anti-human NEDD1 (1:2000) (Proteintech), rabbit anti-human SPAG5 (1:2000) (Proteintech), rabbit anti-human KNTC1 (1:2000) (Proteintech), rabbit anti-human ASPM (1:2000) (Proteintech), rabbit anti-human TUBGCP6 (1:2000) (Novus biologicals), mouse anti-human NPM1 (1:2000) (Proteintech) and mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich).

Techniques: Knockdown, Two Tailed Test, Expressing, Control, Immunostaining, Staining, Marker

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: In wild-type K562 and RPE-1 cells with KIF18A depletion (middle: + KIF18A KD), although KIF18A depletion enhances microtubule dynamics and increases peripheral chromosomes, CENP-E downstream of CENP-C compensates for KIF18A depletion, supporting chromosome congression and alignment, albeit with a delay in the congression. By contrast, in CENP-E-deficient cells (bottom: K562 CENP-C ΔM12BD and RPE-1 CENP-C ΔM12BD cells), KIF18A depletion disrupts chromosome congression due to weakened CENP-E activity downstream of CENP-C at kinetochores, leading to mitotic arrest and subsequent cell death. Based on these observations, we propose that the CENP-C pathway plays a role in chromosome congression, with KIF18A and CENP-E acting cooperatively to promote the congression of peripheral chromosomes during early prometaphase in wild-type K562 and RPE-1 cells (upper).

Article Snippet: The primary antibodies used in this study were, Guinea pig anti-human CENP-C (1:10,000), rabbit anti-Cas9 (1:2,000, Takara Bio), mouse anti-human CENP-A (1:1,000), rabbit anti-human DSN1 (1:5,000), rabbit anti-human KIF18A (1:2000) (Bethyl), rabbit anti-human KIF15 (1:2000) (Proteintech), rabbit anti-human NDE1 (1:2000) (Proteintech), rabbit anti-human NEDD1 (1:2000) (Proteintech), rabbit anti-human SPAG5 (1:2000) (Proteintech), rabbit anti-human KNTC1 (1:2000) (Proteintech), rabbit anti-human ASPM (1:2000) (Proteintech), rabbit anti-human TUBGCP6 (1:2000) (Novus biologicals), mouse anti-human NPM1 (1:2000) (Proteintech) and mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich).

Techniques: Activity Assay