Journal: Oncotarget

Article Title: Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

doi: 10.18632/oncotarget.13206

Figure Lengend Snippet: A. Localization of CENP-E at the midbody of telophase HeLa cells revealed by immunofluorescence staining with anti-CENP-E (green) and anti-α-tubulin (red). DNA was labeled with DAPI (blue). White bar indicates 10 μm. B. CENP-E expression in cytoplasmic, chromatin, and cytoskeletal fractions of HeLa cells. HeLa cells were collected 12 h after release from double-thymidine block and fractionated. Immunoblotting was performed for CENP-E and other mitotic kinesins (Eg5and MKLP2). GAPDH, histone H3, and vimentin were used as cytoplasm, chromatin, and cytoskeleton markers, respectively. C. Immunoblotting of CENP-E and BubR1 in HeLa cells transfected with siCENP-E and siBubR1. HeLa cells were treated with the indicated siRNAs for 24 h. The upper band in the BubR1 rectangle represents phosphorylated (activated) BubR1. GAPDH was used as the gel loading control. D. Representative time-lapse images of HeLa cells transfected with siCENP-E and siBubR1 (lower) or control siNS (upper). Frames were captured at the indicated time points (min) from onset to end of cytokinesis. White arrows indicate the cleavage furrow. White bar indicates 20 μm. E. Effects of CENP-E+BubR1 or BubR1 knockdown alone on cytokinesis duration estimated from time-lapse images (red, siCENP-E+siBubR1 HeLa cells (N = 26); green, siBubR1 HeLa cells (N = 19); blue, siNS HeLa cells (N = 22). Statistical analysis was performed using Student's t-test, and differences were considered significant at p < 0.01.

Article Snippet: The ATPase assay for human CENP-E activity was performed using 6.7 μg/mL of the CENP-E motor domain, 66.7 μg/mL microtubules (Cytoskeleton Inc.), 300 μM ATP, and the indicated GST-fusion proteins (0, 0.5, 1.25, and 2.5 μg).

Techniques: Immunofluorescence, Staining, Labeling, Expressing, Blocking Assay, Western Blot, Transfection

Journal: Oncotarget

Article Title: Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

doi: 10.18632/oncotarget.13206

Figure Lengend Snippet: A. Schematic diagrams of CENP-E fragments. Each fragment contains an HA-tag in the N-terminus. B. Summary of midbody localization by each CENP-E fragment. The ability (+) or inability (−) to localize to the midbody is shown. C. Representative IF images of different HA-tagged CENP-E fragments localized at the midbody. Green, red, and blue signals indicate HA-tagged CENP-E fragments, α-tubulin, and DAPI, respectively. D. Quantified signal intensity of CENP-E fragments at the midbody [IF intensity in a circle of 2-μm radius normalized to the mean intensity of cells transfected with the 2443–2673 (Δ2659–2666) fragment]. Statistical analysis was performed using Student's t-test. Differences were considered significant at p < 0.05. The n.s. indicates “not significant.” E. Immunoblotting of HA in HeLa cells transfected with the indicated HA-tagged CENP-E fragments. The indicated HA-tagged CENP-E fragments were transfected into HeLa cells. Twenty-four hours after transfection, the cells were collected for immunoblotting analysis. GAPDH was used as the gel loading control. All fragments were robustly expressed. F. Fraction (%) of cytokinetic HeLa cells expressing the indicated CENP-E fragments (mean ± standard deviation). Statistical analysis was performed using Student's t-test. Differences were considered significant at p < 0.05. The n.s. indicates “not significant.”

Article Snippet: The ATPase assay for human CENP-E activity was performed using 6.7 μg/mL of the CENP-E motor domain, 66.7 μg/mL microtubules (Cytoskeleton Inc.), 300 μM ATP, and the indicated GST-fusion proteins (0, 0.5, 1.25, and 2.5 μg).

Techniques: Transfection, Western Blot, Expressing, Standard Deviation

Journal: Oncotarget

Article Title: Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

doi: 10.18632/oncotarget.13206

Figure Lengend Snippet: A. The MLS sequence was conjugated to cell-permeable Arg 11 . B. Immunofluorescence of CENP-E in HeLa cells treated with the MSL peptide or control peptide (100 μM) for 24 h. Immunofluorescence staining was performed with anti-CENP-E (red) and anti-α-tubulin (green). DNA was labeled with DAPI (blue). White bar indicates 5 μm. C and D. Quantification of CENP-E immunofluorescence in HeLa cells treated with the MLS peptide or control peptide. The midbody localization of CENP-E was quantified by the IF intensity in a circle of 1.5-μm radius at the midbody (control peptide; N = 48, MLS peptide; N = 56) normalized to the mean intensity of control peptide-treated cells. Statistical analysis was performed using Student's t-test. Differences were considered significant at p < 0.05. The quantified CENP-E signals were categorized into three groups based on the relative IF intensity: high (>1.1), medium (1.1–0.8), and low (<0.8).

Article Snippet: The ATPase assay for human CENP-E activity was performed using 6.7 μg/mL of the CENP-E motor domain, 66.7 μg/mL microtubules (Cytoskeleton Inc.), 300 μM ATP, and the indicated GST-fusion proteins (0, 0.5, 1.25, and 2.5 μg).

Techniques: Sequencing, Immunofluorescence, Staining, Labeling

Journal: Oncotarget

Article Title: Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

doi: 10.18632/oncotarget.13206

Figure Lengend Snippet: A. Schematic of CENP-E fragments consisting of the motor region and MDL (Motor+MLD; upper) or just the MLD (lower). B. Effect of the CENP-E motor domain on midbody localization. Representative IF images of the Motor+MLD and MLD peptides at the midbody. Immunofluorescence was performed with anti-HA (green) and anti α-tubulin (red). DNA was labeled with DAPI (blue). Scale bar, 5 μm. C and D. Effect of CENP-E ATPase activity on midbody localization. ATPase activity of the CENP-E motor domain was attenuated by Cmpd-A (a CENP-E inhibitor) (C) or replacement of amino acids in the ATP-binding pocket of the motor domain (D). Representative images showing CENP-E fragment localization to the midbody. Immunofluorescence was performed with anti-HA (green) and anti-α-tubulin (red). DNA was labeled with DAPI (blue). Scale bars, 10 μm.

Article Snippet: The ATPase assay for human CENP-E activity was performed using 6.7 μg/mL of the CENP-E motor domain, 66.7 μg/mL microtubules (Cytoskeleton Inc.), 300 μM ATP, and the indicated GST-fusion proteins (0, 0.5, 1.25, and 2.5 μg).

Techniques: Immunofluorescence, Labeling, Activity Assay, Binding Assay

Journal: Oncotarget

Article Title: Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

doi: 10.18632/oncotarget.13206

Figure Lengend Snippet: A. Immunofluorescence of CENP-E in HeLa cells treated with Cmpd-A (200 nM) or DMSO for 2 h. Immunofluorescence staining was performed with anti-CENP-E (red) and anti-α-tubulin (green). DNA was labeled with DAPI (blue). White bar indicates 5 μm. B and C. Quantification of CENP-E immunofluorescence in HeLa cells treated with Cmpd-A or DMSO. The midbody localization of CENP-E was quantified by the IF intensity in a circle of 1.5-μm radius at the midbody (DMSO; N = 41, Cmpd-A; N = 43) normalized to the mean intensity of DMSO-treated cells. Statistical analysis was performed using Student's t-test. Differences were considered significant at p < 0.05. The quantified CENP-E signals were categorized into three groups based on the relative IF intensity: high (>1.1), medium (1.1–0.8), and low (<0.8).

Article Snippet: The ATPase assay for human CENP-E activity was performed using 6.7 μg/mL of the CENP-E motor domain, 66.7 μg/mL microtubules (Cytoskeleton Inc.), 300 μM ATP, and the indicated GST-fusion proteins (0, 0.5, 1.25, and 2.5 μg).

Techniques: Immunofluorescence, Staining, Labeling

Journal: Oncotarget

Article Title: Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

doi: 10.18632/oncotarget.13206

Figure Lengend Snippet: A. The CENP-E Motor+MLD fragment colocalized with PRC1 at the midbody. Representative images of Motor+MLD colocalized with PRC1 at the midbody. Immunofluorescence was performed with anti-HA (green) and anti-PRC1 (red). DNA was labeled with DAPI (blue). Histogram of the line scanning indicates the intensity of each signal. B. N- and C-terminal CENP-E fragments interacted with recombinant PRC1 in vitro . Recombinant His-tagged PRC1 proteins and the indicated GST-tagged CENP-E fragment were used for GST pull-down assays (left, GST pull-down; right, 5% input). Immunoblotting with anti-PRC1 or anti-GST antibodies was performed. C. Interaction of PRC1 and endogenous CENP-E. The GST-tagged PRC1 recombinant proteins, microtubules, and cell lysates from 293T cells were used for GST pull-down assays (left, GST pull-down; right, 5% input). Immunoblotting with anti-CENP-E, anti-GST, or anti-α-tubulin antibodies was performed. D. Schematic diagram of in vitro enzymatic assay for CENP-E motor activity. E. PRC1 suppresses CENP-E motor activity in vitro . The X-axis indicates the concentration of GST-PRC1 (red) or GST (blue), and the Y-axis indicates the ATPase activity of the CENP-E motor domain. Statistical analysis was performed using Student's t-test. Differences were considered significant at p < 0.05.

Article Snippet: The ATPase assay for human CENP-E activity was performed using 6.7 μg/mL of the CENP-E motor domain, 66.7 μg/mL microtubules (Cytoskeleton Inc.), 300 μM ATP, and the indicated GST-fusion proteins (0, 0.5, 1.25, and 2.5 μg).

Techniques: Immunofluorescence, Labeling, Recombinant, In Vitro, Western Blot, Enzymatic Assay, Activity Assay, Concentration Assay

Journal: Oncotarget

Article Title: Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

doi: 10.18632/oncotarget.13206

Figure Lengend Snippet: A. Effect of CENP-E motor activity on localization of PRC1 at the midbody. HeLa cells were transfected with CENP-E Motor+MLD. Twenty-four hours after transfection, cells were treated with 200 nM Cmpd-A (lower) or vehicle (DMSO, upper) for 1 h. Following fixation, cells were stained with anti-PRC1 (red) and anti-HA (green). DNA was labeled with DAPI (blue). B. Quantification of PRC1 dispersion (length) at the midbody. The graph shows the distance of PRC1 at the midbody based on immunofluorescence analysis. Statistical analysis was performed using Student's t-test. Differences were considered significant at p < 0.05. C. Correlation of Motor+MLD and PRC1 dispersion (distances) at the midbody. The graph shows the quantitative distance of PRC1 (Y-axis) and the Motor+MLD (X-axis) based on immunofluorescence analysis. D. Quantification of PRC1 immunofluorescence in HeLa cells treated with Cmpd-A or DMSO. The midbody localization of PRC1 was quantified by the IF intensity in a circle of 1.5-μm radius at the midbody (DMSO; N = 31, Cmpd-A; N = 36) normalized to the mean intensity of DMSO-treated cells. Statistical analysis was performed using Student's t-test. Differences were considered significant at p < 0.05. E . Quantification of PRC1 immunofluorescence in siBubR1-transfected HeLa cells treated with Cmpd-A or DMSO. Twenty-four hours after siBubR1 transfection, HeLa cells were treated with 200 nM Cmpd-A or DMSO for 24 hours. The midbody localization of PRC1 was quantified by the IF intensity in a circle of 1.5-μm radius at the midbody (DMSO; N = 30, Cmpd-A; N = 30) normalized to the mean intensity of DMSO-treated cells. Statistical analysis was performed using Student's t-test. Differences were considered significant at p < 0.05.

Article Snippet: The ATPase assay for human CENP-E activity was performed using 6.7 μg/mL of the CENP-E motor domain, 66.7 μg/mL microtubules (Cytoskeleton Inc.), 300 μM ATP, and the indicated GST-fusion proteins (0, 0.5, 1.25, and 2.5 μg).

Techniques: Activity Assay, Transfection, Staining, Labeling, Immunofluorescence

Journal: Oncotarget

Article Title: Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

doi: 10.18632/oncotarget.13206

Figure Lengend Snippet: A. PRC1 knockdown attenuated MLD localization to the midbody. HeLa cells were transfected with the MLD plasmid of CENP-E 24 h after siNS (upper) or siPRC1 (lower) treatment. Twenty-four hours after transfection, cells were fixed for immunofluorescence staining with anti-HA (green) and anti-PRC1 (red). DNA was labeled with DAPI (blue). B. Fraction (%) of HeLa cells expressing the indicated CENP-E fragments during cytokinesis in the presence of siNS (blue) or siPRC1 (red). C. Fraction (%) of multinucleated HeLa cells expressing the indicated CENP-E fragments in the presence of siNS (blue) or siPRC1 (red). D. Schematic of CENP-E motor activity associated with the PRC1 localization at the midbody.

Article Snippet: The ATPase assay for human CENP-E activity was performed using 6.7 μg/mL of the CENP-E motor domain, 66.7 μg/mL microtubules (Cytoskeleton Inc.), 300 μM ATP, and the indicated GST-fusion proteins (0, 0.5, 1.25, and 2.5 μg).

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining, Labeling, Expressing, Activity Assay