Journal: Cancers

Article Title: Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

doi: 10.3390/cancers15204911

Figure Lengend Snippet: Figure 1. An in silico bioinformatics screen identifies mitotic proteins essential for EWS growth. (A) The DepMap portal was used to access the RNA expression data across different cancer cell lines. Expression in Ewing sarcoma is highlighted in red. Capillary-based analysis of protein lysates from EWS cell lines indicating expression of (B) KIF11 and AURKA and (C) KIF15 and TPX2 protein levels. The uncropped blots are shown in Figure S3.

Article Snippet: After this denaturation step, the prepared samples; blocking reagent; 1:50 diluted primary antibodies KIF11 or Eg5 (Cell Signaling Technology, Danvers, MA, USA, Cat# 4203, RRID:AB_10545760), p-KIF11 (Thermo Fisher Scientific Cat# PA5-105186, RRID:AB_2816659), KIF15 (Proteintech, Rosemont, IL, USA, Cat# 55407-1- AP, RRID:AB_11182836), AURKA (Novus Cat# NBP1-51843SS, RRID:AB_11018019), pAURKA (Novus Cat# NBP3-05434) PARP(46D11) (Cell Signaling Technology Cat# 9532, RRID:AB_659884), and β-actin (Cell Signaling Technology Cat# 12262, RRID:AB_2566811); HRP-conjugated secondary antibodies anti-rabbit (Biotechne, Minneapolis, MN, USA, #DM-001) and anti-mouse (Biotechne, #DM-002); and chemiluminescent substrate were dispensed into designated wells in an assay plate.

Techniques: In Silico, RNA Expression, Expressing

Journal: Cancers

Article Title: Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

doi: 10.3390/cancers15204911

Figure Lengend Snippet: Figure 2. Synergistic inhibition of EWS growth by VIC-1911 and different KIF11 inhibitors. Dose– response curves, dose–response matrix, and heat map indicating synergy scores in TC-71 EWS cell

Article Snippet: After this denaturation step, the prepared samples; blocking reagent; 1:50 diluted primary antibodies KIF11 or Eg5 (Cell Signaling Technology, Danvers, MA, USA, Cat# 4203, RRID:AB_10545760), p-KIF11 (Thermo Fisher Scientific Cat# PA5-105186, RRID:AB_2816659), KIF15 (Proteintech, Rosemont, IL, USA, Cat# 55407-1- AP, RRID:AB_11182836), AURKA (Novus Cat# NBP1-51843SS, RRID:AB_11018019), pAURKA (Novus Cat# NBP3-05434) PARP(46D11) (Cell Signaling Technology Cat# 9532, RRID:AB_659884), and β-actin (Cell Signaling Technology Cat# 12262, RRID:AB_2566811); HRP-conjugated secondary antibodies anti-rabbit (Biotechne, Minneapolis, MN, USA, #DM-001) and anti-mouse (Biotechne, #DM-002); and chemiluminescent substrate were dispensed into designated wells in an assay plate.

Techniques: Inhibition

Journal: Cancers

Article Title: Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

doi: 10.3390/cancers15204911

Figure Lengend Snippet: Figure 6. Analysis of protein expression post-drug treatment. (A) CHLA-10 and (B) TC-71 cells treated with drugs were assessed for changes in protein expression 24 h post-treatment via capillary electrophoresis-based Wes analysis. Increased protein levels of KIF11, p-KIF11Thr926 AURKA, and p-AURKAThr288 were observed for the drug combination group, whereas KIF15 levels were noticeably lower. Similarly, enhanced cleaved-PARP expression was observed with the combination treatment. The uncropped blots are shown in Figures S8 and S9.

Article Snippet: After this denaturation step, the prepared samples; blocking reagent; 1:50 diluted primary antibodies KIF11 or Eg5 (Cell Signaling Technology, Danvers, MA, USA, Cat# 4203, RRID:AB_10545760), p-KIF11 (Thermo Fisher Scientific Cat# PA5-105186, RRID:AB_2816659), KIF15 (Proteintech, Rosemont, IL, USA, Cat# 55407-1- AP, RRID:AB_11182836), AURKA (Novus Cat# NBP1-51843SS, RRID:AB_11018019), pAURKA (Novus Cat# NBP3-05434) PARP(46D11) (Cell Signaling Technology Cat# 9532, RRID:AB_659884), and β-actin (Cell Signaling Technology Cat# 12262, RRID:AB_2566811); HRP-conjugated secondary antibodies anti-rabbit (Biotechne, Minneapolis, MN, USA, #DM-001) and anti-mouse (Biotechne, #DM-002); and chemiluminescent substrate were dispensed into designated wells in an assay plate.

Techniques: Expressing, Electrophoresis

Journal: Scientific Reports

Article Title: Overexpression of the NEK9–EG5 axis is a novel metastatic marker in pathologic stage T3 colon cancer

doi: 10.1038/s41598-022-26249-0

Figure Lengend Snippet: Univariate and multivariate regression analysis of metastasis biomarkers in the study cohort.

Article Snippet: The samples were incubated with each of the following primary antibodies: NEK9 (1:500; ab138488, Abcam, UK), EG5 (1:100; 4203, Cell Signaling Technology, Boston, MA), acetyl-α-tubulin (1:1000; 5335, Cell Signaling Technology), E-cadherin (1:200; clone 4A2C7, Zymed, CA), claudin-1 (1:100; #359A-15, Cell Marque, CA ), vimentin (1:500, clone V9, Zymed), and ꞵ-catenin (1:200, clone 14, Cell Marque).

Techniques:

Journal: Scientific Reports

Article Title: Overexpression of the NEK9–EG5 axis is a novel metastatic marker in pathologic stage T3 colon cancer

doi: 10.1038/s41598-022-26249-0

Figure Lengend Snippet: Correlation between NEK9, EG5, and acetyl-α-tubulin immunostaining in the study cohort.

Article Snippet: The samples were incubated with each of the following primary antibodies: NEK9 (1:500; ab138488, Abcam, UK), EG5 (1:100; 4203, Cell Signaling Technology, Boston, MA), acetyl-α-tubulin (1:1000; 5335, Cell Signaling Technology), E-cadherin (1:200; clone 4A2C7, Zymed, CA), claudin-1 (1:100; #359A-15, Cell Marque, CA ), vimentin (1:500, clone V9, Zymed), and ꞵ-catenin (1:200, clone 14, Cell Marque).

Techniques: Immunostaining

Journal: Scientific Reports

Article Title: Overexpression of the NEK9–EG5 axis is a novel metastatic marker in pathologic stage T3 colon cancer

doi: 10.1038/s41598-022-26249-0

Figure Lengend Snippet: Concordant expression of the NEK9–G5 axis during the G 2 /M phase of the cell cycle. ( a ) Cell cycle distribution of SW480 and SW620 colon cancer cells treated with nocodazole (500 nM) for 24 or 48 h and then released from this block. ( b ) Western blot analysis of phosphorylated NKE9 (pNEK9 T210 ), NEK9, EG5, acetyl-tubulin, phosphorylated AKT (pAKT S473 ) and cyclin B1 at the indicated times of nocodazole–induced G 2 /M arrest, and after the release from this block.

Article Snippet: The samples were incubated with each of the following primary antibodies: NEK9 (1:500; ab138488, Abcam, UK), EG5 (1:100; 4203, Cell Signaling Technology, Boston, MA), acetyl-α-tubulin (1:1000; 5335, Cell Signaling Technology), E-cadherin (1:200; clone 4A2C7, Zymed, CA), claudin-1 (1:100; #359A-15, Cell Marque, CA ), vimentin (1:500, clone V9, Zymed), and ꞵ-catenin (1:200, clone 14, Cell Marque).

Techniques: Expressing, Blocking Assay, Western Blot

Journal: Scientific Reports

Article Title: Overexpression of the NEK9–EG5 axis is a novel metastatic marker in pathologic stage T3 colon cancer

doi: 10.1038/s41598-022-26249-0

Figure Lengend Snippet: Kaplan–Meier survival curves for overall survival (OS) according to the expression of NEK9, EG5 or acetyl-α-tubulin. ( a ) NEK9 expression in the whole cohort (n = 136). ( b ) NEK9 expression in the M0 patient subgroup (stage III; n = 87). ( c ) EG5 expression in the whole cohort. ( d ) Acetyl-α-tubulin expression in the whole cohort. OS differences were observed between the low- and high-expression cases for these proteins, with a more distinct difference for NEK9. M0, patients with no evidence of metastasis.

Article Snippet: The samples were incubated with each of the following primary antibodies: NEK9 (1:500; ab138488, Abcam, UK), EG5 (1:100; 4203, Cell Signaling Technology, Boston, MA), acetyl-α-tubulin (1:1000; 5335, Cell Signaling Technology), E-cadherin (1:200; clone 4A2C7, Zymed, CA), claudin-1 (1:100; #359A-15, Cell Marque, CA ), vimentin (1:500, clone V9, Zymed), and ꞵ-catenin (1:200, clone 14, Cell Marque).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Overexpression of the NEK9–EG5 axis is a novel metastatic marker in pathologic stage T3 colon cancer

doi: 10.1038/s41598-022-26249-0

Figure Lengend Snippet: Immunohistochemical expression of NEK9, EG5 and acetyl-α-tubulin and clinicopathologic characteristics.

Article Snippet: The samples were incubated with each of the following primary antibodies: NEK9 (1:500; ab138488, Abcam, UK), EG5 (1:100; 4203, Cell Signaling Technology, Boston, MA), acetyl-α-tubulin (1:1000; 5335, Cell Signaling Technology), E-cadherin (1:200; clone 4A2C7, Zymed, CA), claudin-1 (1:100; #359A-15, Cell Marque, CA ), vimentin (1:500, clone V9, Zymed), and ꞵ-catenin (1:200, clone 14, Cell Marque).

Techniques: Immunohistochemical staining, Expressing

Journal: International Journal of Biological Sciences

Article Title: The Interaction of Calcium-Sensing Receptor with KIF11 Enhances Cisplatin Resistance in Lung Adenocarcinoma via BRCA1/cyclin B1 pathway

doi: 10.7150/ijbs.92046

Figure Lengend Snippet: KIF11 interacted with CaSR and was upregulated in DDP-resistant LUAD cells. Mass spectrometry (MS) was used to identify proteins (A-C) , CaSR-interacting proteins (A) , KIF11-interacting proteins (B) , and proteins interacting with CaSR or KIF11 are selected based on two screening criteria: The protein was not pulled down by IgG or the protein had less than 2 folds increase in detection in IgG compared to CaSR or KIF11 (C) . Protein-protein interaction maps between CaSR and KIF11 (D) , CaSR is depicted in dark blue, and KIF11 is depicted in cyan. The corresponding binding points are indicated as clubbed structures of the corresponding colors. And western blotting results showed clear detection of KIF11 in the CaSR-interacting proteins (E) and CaSR in the KIF11-interacting proteins (F) . KIF11 was highly expressed in LUAD (G) . Kaplan-Meier plots showed the correlation of KIF11 with overall survival in LUAD (H) , including the patient underwent chemotherapy (I) .

Article Snippet: A kinesin family member 11 (KIF11) inhibitor (EG5 Inhibitor V, trans-24; T11155) was purchased from TargetMol (Boston, MA, USA).

Techniques: Mass Spectrometry, Binding Assay, Western Blot

Journal: International Journal of Biological Sciences

Article Title: The Interaction of Calcium-Sensing Receptor with KIF11 Enhances Cisplatin Resistance in Lung Adenocarcinoma via BRCA1/cyclin B1 pathway

doi: 10.7150/ijbs.92046

Figure Lengend Snippet: Downregulation of KIF11 inhibited the growth of DDP-resistant LUAD cells. A549-DDP and H1299-DDP cells were transfected with siRNA targeting KIF11 (siKIF11-1, siKIF11-2) or non-targeting siRNA (si-NC). After KIF11 knockdown, the clonogenic proliferation of cisplatin-resistant cells was disrupted (A) . The proliferation ability of A549-DDP and H1299-DDP were inhibited and the panels show the relative cell viability at the 48 h (B) . Western blotting results showed the changes on protein levels of KIF11, CaSR, cyclin B1, CDK1 and BRCA1 (C) . Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns, not significant.

Article Snippet: A kinesin family member 11 (KIF11) inhibitor (EG5 Inhibitor V, trans-24; T11155) was purchased from TargetMol (Boston, MA, USA).

Techniques: Transfection, Knockdown, Western Blot

Journal: International Journal of Biological Sciences

Article Title: The Interaction of Calcium-Sensing Receptor with KIF11 Enhances Cisplatin Resistance in Lung Adenocarcinoma via BRCA1/cyclin B1 pathway

doi: 10.7150/ijbs.92046

Figure Lengend Snippet: Inhibition of KIF11 reduced cisplatin resistance in DDP-resistant LUAD cells. The clonogenic proliferation of DDP-resistant cells was disrupted by the KIF11 inhibitor (A) . A549-DDP cells and H1299-DDP cells were treated with 10 µM cisplatin and 1µM KIF11 inhibitor, respectively, or in combination. Colony formation assay was performed to analyze the cells, and the right panel quantifies the relative number of colonies formed after 14 days. The sensitivity of A549-DDP (B and C) and H1299-DDP (D and E) cells to cisplatin was affected by the KIF11 inhibitor. The protein levels of KIF11, CaSR, cyclin B1, CDK1 and BRCA1 were detected by western blotting (F) . The schematics illustrate the proposed mechanism of CaSR activates the cisplatin resistance in LUAD (G) . Data are showed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns, not significant.

Article Snippet: A kinesin family member 11 (KIF11) inhibitor (EG5 Inhibitor V, trans-24; T11155) was purchased from TargetMol (Boston, MA, USA).

Techniques: Inhibition, Colony Assay, Western Blot

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet: Depletion of PRC1 and inactivation of EG5 block spindle elongation during anaphase (A) Two models of spindle elongation. (B) STED image (single z plane) of an anaphase spindle in a live U2OS cell expressing CENP-A-GFP (magenta). Microtubules are labeled with 100-nM SiR-tubulin (green). Arrowheads point to the spindle midzone where antiparallel microtubules are located. (C) Live-cell images (top) and corresponding kymographs (bottom) of control, STLC-treated (EG5 inh.), PRC1-siRNA-depleted (indicated by an arrow pointing down) and PRC1-siRNA-depleted 40-μM STLC-treated RPE-1 cells stably expressing CENP-A-GFP and centrin1-GFP. kin, kinetochore; cen, centrosome. White arrowheads indicate centrioles. Horizontal gray lines in the kymographs indicate the onset of anaphase. (D) Quantification of spindle elongation velocity (see scheme) (n = 34, 16, 10, 9, 17, 10, 15, 6, 7, 8, 11, 7, and 10 cells, from left to right), measured in the period from the 1–3 min after anaphase onset for each treatment. More than three independent experiments for every condition regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Boxes represent standard deviation (dark gray), 95% confidence interval of the mean (light gray), and mean value (black). Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (C). Statistics: t test ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). Time is shown as minutes. Images are the maximum projection of the acquired z stack. Time 0 represents anaphase onset. Horizontal scale bars, 1 μm. Vertical scale bars, 1 min. See also Figure S1 .

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Blocking Assay, Expressing, Labeling, Control, Stable Transfection, Standard Deviation

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet: Chromosome segregation is compromised after KO of PRC1 and inhibition of EG5 (A) Live images of RPE-1 cells in PRC1 non-induced KO, induced PRC1 CRISPR KO and induced PRC1 KO treated with 40-μM STLC imaged 4 days after doxycycline induction. 100-nM SiR-DNA (cyan) was used for chromosome staining. (B) Quantification of chromosome segregation velocity (see scheme) in CRISPR experiments (n = 14, 11, and 11, from left to right). Two independent experiments for every condition except PRC1 KO + Eg5 inh. which was done in three independent experiments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (A). Statistics: t test ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (C) Live-cell images of induced RPE-1 PRC1 CRISPR KO and induced PRC1 KO treated with 40-μM STLC. Dashed lines designate the cell borders. 100-nM SiR-DNA was used for chromosome staining. Time is shown as minutes. Images are maximum projections of the acquired z stack. Time 0 represents anaphase onset. Scale bars, 1 μm. See also Figure S2 .

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Inhibition, CRISPR, Staining

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet: Depletion of KIF4A and inactivation of EG5 induce a block in chromosome segregation by blocking spindle elongation (A) Live-cell images of control, KIF4A siRNA-depleted, KIF4A-siRNA-depleted 40-μM STLC-treated, and KIF4A-siRNA-depleted 100-μM monastrol-treated RPE-1 cells stably expressing CENP-A-GFP and centrin1-GFP. kin, kinetochore; cen, centrosome. White arrowheads indicate centrioles. Horizontal gray bars in the kymographs indicate the onset of anaphase. Time zero represents anaphase onset. Time is shown as minutes. (B) Quantification of velocity of spindle elongation (n = 34, 10, 15, 8, 10, 12, 7, 6, 11, and 10 cells, from left to right). Three independent experiments for every condition regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (A). Statistics: t test ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (C) Live images of KIF4A siRNA-depleted STLC-treated RPE-1 cell show complete failure in chromosome segregation after STLC addition during early anaphase. Time zero represents the start of STLC treatment. White circles represent the locations of the initiation of the cytokinetic cleavage furrowing. Time is shown as minutes:seconds. Images are the maximum projection of the acquired z stack. Horizontal scale bars, 1 μm. Vertical scale bars, 1 min. See also Figure S3 .

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Blocking Assay, Control, Stable Transfection, Expressing

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet: Depletion of both kinesin-6 MKLP1 and kinesin-6 MKLP2 with inactivation of EG5 induces a partial block in chromosome segregation by disrupting spindle elongation (A) Live-cell images of control, MKLP1- and MKLP2-siRNA co-depleted and MKLP1- and MKLP2 siRNA co-depleted 40-μM STLC-treated RPE-1 cells stably expressing CENP-A-GFP and centrin1-GFP. kin, kinetochore; cen, centrosome. White arrowheads indicate centrioles. Horizontal gray bars in the kymographs indicate the onset of anaphase. (B) Quantification of velocity of spindle elongation (n = 34, 17, 12, and 11 cells, from left to right). Three independent experiments for every condition regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (A). Statistics: t test ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (C) Quantification of net spindle elongation from anaphase onset to 5-min afterward for treatments as indicated (n = 7, 10, 9, and 10 cells, from left to right). Time is shown as minutes. Images are the maximum projection of the acquired z stack. Time 0 represents anaphase onset. Horizontal scale bars, 1 μm. Vertical scale bars, 1 min. (D) Immunofluorescence images of fixed RPE-1 cells depleted of target proteins by treatment with indicated siRNAs stably expressing CENP-A-GFP and centrin1-GFP (magenta) stained with AlexaFluor594 conjugated with antibody specific to the indicated target protein (green KIF4A and red MKLP2). Chromosomes were stained with 1 μg/mL DAPI solution (cyan). Scale bars, 2 μm (D). (E) Quantification of mean signal intensity measured in the spindle midzone of KIF4A (top) and MKLP2 (bottom) antibodies conjugated with AlexaFluor594 versus spindle length in the indicated conditions (8 MKLP2 depleted and 5 control cells). Two independent experiments for every condition regarding siRNAs or non-targeting treatments. See also Figure S4 .

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Blocking Assay, Control, Stable Transfection, Expressing, Immunofluorescence, Staining

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet: Decreased midzone stability does not affect chromosome segregation velocity during anaphase (A and B) Immunofluorescent (A) and expansion microscopy (B) images of fixed control, PRC1-siRNA-depleted (only immunofluorescence) and KIF4A-siRNA-depleted 40-μM STLC-treated RPE-1 cells stained with AlexaFluor594 conjugated with α-tubulin antibody. Images in (A) and (B) are the maximum projections of color-coded z stacks as shown by the scheme in (B). Arrowheads point to the spindle midzone region. (C) STED images (single z plane) of anaphase spindles labeled with 100 nM SiR-tubulin in live non-induced CRISPR PRC1 KO and induced PRC1 KO RPE-1 cells. (D) Smoothed live-cell images (single z plane) of RPE-1 cells after midzone photoactivation of photoactivatable (PA)-GFP-α-tubulin (green) in the indicated conditions. 100-nM SiR-DNA (magenta) was used for chromosome staining. Time 0 represents the photoactivation onset. (E) Quantification of midzone stability, defined as the ratio of the integrated photoactivated signal in the boxed region (see schemes and boxes in D) at times 30 and 0 s, and chromosome segregation velocity measured in the same time period (see schemes), for the indicated treatments. Statistics: t test ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). Numbers: 14, 7, 8, 9, 6, 5, 15, and 5 cells, from left to right. Three independent experiments for every condition except MKLP1 + MKLP2, MKLP1 + MKLP2 + EG5 inh. and KIF4+EG5 inh., which were done in two independent experiments regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (D). (F) Chromosome segregation velocity versus midzone stability (data from E) for indicated treatments and linear regression (line); r s , Spearman correlation coefficient, p < 0.001. Scale bars, 1 μm. See also Figure S5 .

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Microscopy, Control, Immunofluorescence, Staining, Labeling, CRISPR

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet: KIF4A depletion and EG5 inhibition does not impact the number or dynamic properties of midzone and astral microtubules (A) Live-cell images of control RPE-1 cells expressing EB3-eGFP and H2B-mCherry showing schematically the definition of two astral regions, and the midzone region between H2B-labeled chromosomes (top) and the same cell with tracked EB3 spots (magenta circles) using TrackMate ImageJ tool (bottom). (B) Live-cell images of RPE-1 cells expressing EB3-eGFP in control, KIF4A siRNA depletion and KIF4A siRNA depletion and 40-μM STLC treatment in early anaphase (top row) and late anaphase (bottom row). Images are maximum temporal projections of color-coded time points as shown on the scheme generated from a total of 1 min of early and late anaphase time frames. (C) Live-cell images (top) and corresponding schemes (bottom) of RPE-1 cells in mid anaphase (3 min from the onset) and telophase (15 min from the onset) after KIF4A depletion and EG5 inhibition. Dashed lines indicate the cell borders. (D) Graphs depicting change in the number of EB3 spots in astral regions (first), number of EB3 spots contacting the cortical area (second), number of EB3 spots per micron of midzone (third), and ratio of the number of EB3 spots in astral versus midzone arrays (fourth) in time in both control (n = 12 cells from two independent experiments) and KIF4A-depleted STLC-treated spindles (n = 6 cells from two independent experiments). Shaded areas represent the 95% confidence interval of the mean and thick lines represent the mean values for both indicated treatments. (E) Whole spindle kymographs of EB3-GFP (gray) and H2B-mCherry-expressing (magenta) cells during 10 min of anaphase in the indicated treatments. (F) Quantification of EB3 comet velocity (left) and EB3 comet track length (right) in the indicated treatments (n = 3 cells for KIF4A depletion from two independent experiments, other treatments are the same as in D; the number of comet traces is 80, 35, and 28 from left to right). Statistics: t test. Horizontal scale bars, 5 μm (in E, 1 μm). Vertical scale bar, 1 min. See also Figure S6 .

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Inhibition, Control, Expressing, Labeling, Generated

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet: Chromosome segregation and spindle elongation are dependent upon KIF4A and EG5-generated sliding (A) Montage time-lapse live images of the mitotic spindle midzone region after photoactivation of photoactivatable (PA)-GFP-α-tubulin in the indicated treatments. Top and bottom schemes depict sliding of the photoactivated midzone region. Vertical scale bar, 1 s. Horizontal scale bar, 1 μm. (B) Quantification of sliding velocity, measured as the length change of the photoactivated spot (L, see scheme) in the indicated conditions. Statistics: t test ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). Numbers: 13, 8, 6, 10, 6, 6, 15, and 6 cells, from left to right. Three independent experiments for every condition except MKLP1 + MKLP2 siRNAs and MKLP1 + MKLP2 + EG5 inh., which were done in two independent experiments regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (A and B). (C and D) Chromosome segregation velocity (C) and centrosome separation velocity (D) versus sliding velocity in the indicated treatments, and linear regression (line); r s , Spearman correlation coefficient, p < 0.001. (E) Proposed model for the motor-driven sliding in the antiparallel midzone region (top scheme) of an anaphase mitotic spindle including two independent sliding modules. Gray arrows point to the direction of overall microtubule motion as a result of forces produced by the motors walking on antiparallel microtubules (black arrows). See also Figure S7 .

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Generated, Produced

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Recombinant, Saline, Protease Inhibitor, Transfection, CRISPR, Knock-Out, Control, Plasmid Preparation, Software

Journal: The Journal of Cell Biology

Article Title: Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes

doi: 10.1083/jcb.201410001

Figure Lengend Snippet: Bipolar spindle formation is delayed in Mio-depleted cells. (A) Control and Mio-depleted cells were arrested with Eg5 inhibitor Monastrol for 3 h. The drug was washed out with fresh medium, and cells were fixed at the indicated time points. Cells were immunostained with α-tubulin (red) and α-ACA (green). (B) Quantitation of spindle and chromosome alignment status in control and Mio-depleted cells at the indicated time points after Monastrol release. 300 cells/time point ( n = 3). Bar, 10 µm.

Article Snippet: The other antibodies used are tyrosinated tubulin YL12 (rat; Abcam), α-tubulin (mouse, DMIA; Sigma-Aldrich), PLK1 (mouse; EMD Millipore), p-Plk1T210 (mouse; Abcam), Aurora A (mouse; BD), P–Aurora A T288 (rabbit; Abcam), Eg5 (rabbit; Novus Biologicals), GFP (rabbit; Life Technologies), ACA (human; Antibodies Incorporated), mAb414 (mouse; Covance), pericentrin (rabbit; Abcam), γ-tubulin (mouse; Sigma-Aldrich), Hurp (rabbit; Abcam), Numa (rabbit; Abcam), TPX2 (mouse; Abcam), p150 (mouse; BD), Aurora B (rabbit; Abcam), Survivin (rabbit; Novus Biologicals), Bub1 (mouse; Abcam), Sgo1 (mouse; Abcam), mTOR and p-mTOR2481 (rabbit; Cell Signaling Technology), p70S6K (Thr389, antibody kit, rabbit; Cell Signaling Technology), PTEN (rabbit, D4.3; Cell Signaling Technology), 4EBP1 (53H11, rabbit; Cell Signaling Technology), 4EBP1 (Ser65, rabbit; Cell Signaling Technology), MPM2 (mouse; EMD Millipore), PPP6C (rabbit; Bethyl Laboratories, Inc.), SAPK/JNK (56G8, rabbit; Cell Signaling Technology), SAPK/JNK (Thr183/Tyr185, rabbit; Cell Signaling Technology), p38 MAPK (D13E1, rabbit; Cell Signaling Technology), p38 MAPK (Thr180/Tyr182; rabbit, Cell Signaling Technology), p44/42 MAPK and p44/42 Thr202/Tyr204 (137F5, rabbit; Cell Signaling Technology), and Seh1 (rabbit, generated against full-length histidine-tagged human protein; ).

Techniques: Control, Quantitation Assay

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5

doi: 10.1002/cm.21380

Figure Lengend Snippet: A. Two-color Western blot showing endogenous Eg5 immunoprecipitated from HEK293T or LLC-Pk1 cell lysates, with each channel displayed separately in black and white. A-419259 was added as indicated; the lower panel shows a β-tubulin loading control (green). B. The structure of Eg5 bound to S-trityl-L-cysteine (STLC) is marked with tyrosines Y125, Y211, and Y231 (orange space fill, PDB: 3KEN). A predicted SH3 binding site in the MT-binding site of the Eg5 motor domain is shown in the inset. L5 is shown in dark blue; Loop 12 within the MT binding domain is shown in red. C. Sequence alignment comparing the putative phosphorylation sites and the –PXXP– SH3 targeting domain. Putative phosphorylated tyrosines and the –PXXP– SH3 targeting motifs are shown in red. The accession numbers for each protein are listed in Fig. S1 C.

Article Snippet: For detection of endogenous Eg5 we used 1:5000 polyclonal rabbit anti-Eg5 NB500-181 antibody (Novus Biologicals, Littleton, CO).

Techniques: Western Blot, Immunoprecipitation, Control, Binding Assay, Sequencing, Phospho-proteomics

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5

doi: 10.1002/cm.21380

Figure Lengend Snippet: Kinesin-5 constructs (denoted across the top) were incubated with human c-Src (A) or human Wee1 kinase (B) and radiolabeled ATP for the times indicated. Reactions were quenched, run on an SDS-PAGE (top) that was then dried and exposed to film (bottom). Position of c-Src, Eg5, and Wee1 marked on the left side. C. Immunoprecipitation of Eg5 from HEK293T cells co-transfected with myc-tagged Eg5 motor head and c-Src constructs; A-419259 added as indicated. Western blot stained using anti-myc and anti-pTyr antibodies which here are displayed separately in black and white. The lower panel shows inputs and β-tubulin level as a loading control. D. Endogenous Eg5 was immunoprecipitated from HEK293T cells transfected with the indicated constructs; A-419259 was added as indicated. pTyr was detected using a two-color Western blot. Each channel is displayed separately in black and white. Transfection efficacy was verified by detection of c-Src in the lysates from transfected cells. β-catenin level is shown as a loading control.

Article Snippet: For detection of endogenous Eg5 we used 1:5000 polyclonal rabbit anti-Eg5 NB500-181 antibody (Novus Biologicals, Littleton, CO).

Techniques: Construct, Incubation, SDS Page, Immunoprecipitation, Transfection, Western Blot, Staining, Control

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5

doi: 10.1002/cm.21380

Figure Lengend Snippet: Effects of phosphomimetic and non-phosphorylatable mutations on Eg5 motor characteristics and STLC binding Steady-state ATPase rates and MT sliding velocities of Eg5 phosphomimetic (E) and non-phosphorylatable (F) mutants were measured and compared to those of Eg5-367-WT and Eg5-367-DL5 mutants using standard in vitro assays (Methods). Dissociation constants of the L5 inhibitor STLC to Eg5-367 phosphomimetic and non-phosphorylatable mutants (K D ) was calculated from ITC titrations as described in the Methods. Errors in K D were estimated based on the nonlinear least-squares fits to raw ITC data. Stoichiometries (N) show some variability reflecting protein concentration determination, but are generally consistent with single-site binding.

Article Snippet: For detection of endogenous Eg5 we used 1:5000 polyclonal rabbit anti-Eg5 NB500-181 antibody (Novus Biologicals, Littleton, CO).

Techniques: Binding Assay, In Vitro, Protein Concentration

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5

doi: 10.1002/cm.21380

Figure Lengend Snippet: A. Percent of mitotic phenotypes in LLC-Pk1 cells transfected with siRNA targeting endogenous Eg5 alone or co-transfected with an siRNA resistant Eg5 Emerald construct (WT, Y211E, Y211F, GSTY). Monopole (yellow), bipole (red), muitipole (blue), disorganized (green). Examples of each phenotype are shown on right. B. Time-lapse imaging of LLC-Pk1 cells expressing mCherry-α-tubulin (right panels) co-transfected with Eg5 siRNA and siRNA resistant Eg5 Emerald constructs (WT, Y211E, and Y211F left panels). C. Immunofluorescence staining for MTs in control (left) and SU6656-treated (right) parental LLC-Pk1 cells. D. Quantification of mitotic spindle phenotypes shown in C. E. Immunofluorescence staining for MTs (top) and phospho-Aurora (bottom) for control and SU6656 treated cells. (F) Immunofluorescence staining of α-tubulin in anaphase LLC-Pk1 cells: control (top), BI-2536 (middle), SU6656 (bottom). ** = p ≤ 0.01. Scale bars in A, B, C, E, F = 5 μm. Time in B (min:sec). Error Bars = St Dev.

Article Snippet: For detection of endogenous Eg5 we used 1:5000 polyclonal rabbit anti-Eg5 NB500-181 antibody (Novus Biologicals, Littleton, CO).

Techniques: Transfection, Construct, Imaging, Expressing, Immunofluorescence, Staining, Control