Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: Cytotoxicity of SB743921 against mitotic inhibitors in cells with the wild type (WT) and knocked out p53 ( TP53-/- ). A , IC50 values (mean ± SD, n=3) of A549, MCF7 and HCT116 exposed to various mitotic inhibitors for 7 days were obtained based on the SRB assay. B , Proliferative and colony formation ability of A549 WT and A549 TP53-/- after 7 d or 14 d from the removal of SB743921 (SB) or Ixabepilone (Ixa). Cells were exposed to drugs for 72 h and then incubated in fresh media the following 7 d or 14 d. C, Cell cycle distribution of A549 WT and A549 TP53-/- ( left panels ) and MCF7 WT and MCF7 TP53-/- ( right panels ) treated with SB (10 nM) or Ixa (25 nM) for 24 h ( upper panels ) and 72 h ( bottom panels ).

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Sulforhodamine B Assay, Incubation

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: p53-mediated cell cycle inhibitory and proapoptotic pathways are activated in response to the exposure of SB743921 (SB) or Ixabepilone (Ixa). A, Immunoblotting analysis of MCF7 and A549 treated with SB or Ixa in different concentrations (SB 0.5 nM and 10 nM, Ixa 2 nM and 25 nM) for 24 h. B, C , qPCR analysis of MCF7 and A549 respectively treated with SB (10 nM for MCF7, 3 nM for A549) or Ixa (25 nM) for 16 h (A549) or 24 h (MCF7).

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: SB743921 induces irreversible proliferative arrest and predominant death of A549 TP53-/- cells. A , B , Cell cycle distribution after 6 days after removal of SB743921 (SB) or Ixabepilone (Ixa). Cells were exposed to the drugs for 24 h, washed and incubated for 3 or 6 d in fresh media. C , Confocal microscopy images of EdU-treated samples after a week since the removal of SB or Ixa. Cells were exposed to drugs for 72 h before the washout. D , Single cell concentrations in flow cytometry analysis as a percentage of the untreated control samples. *, P < 0.05, **, P < 0.01; ns – not significant.

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Incubation, Confocal Microscopy, Single Cell, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: SB743921 depletes pro-replicative signaling causing irreversible arrest, while p53 is crucial for G1/S arrest in WT cells. A , Immunoblotting analysis in A549 WT and A549 TP53-/- cells 6 days after removal of SB743921 (SB) or Ixabepilone (Ixa). Cells were exposed to drugs for 24 h, then washed and incubated for 6 d in fresh media. C , qPCR analysis of A549 WT and A549 TP53-/- expression after 6 days since the removal of SB or Ixa. Cells were exposed to drugs for 24 h before the washout.

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Western Blot, Incubation, Expressing

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: SB743921 induces irreversible senescence in A549 WT and SB743921-treated senescent cells can be targeted by senolytics, such as ABT-263. A , Light microscopy images of A549 WT 6 days after the removal of SB743921 (SB) or Ixabepilone (Ixa). Cells were fixed and then stained with solution for the SA-β-Gal detection. B, Quantitative analysis of stained A549 WT and A549 TP53-/- cells as demonstrated in A . Data were compared using the ANOVA test; **, P < 0.01, ****, P < 0.0001; ns – not significant. C, Crystal violet staining of A549 WT and A549 TP53-/- treated with SB or Ixa alone or together with 3 uM ABT-263.

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Light Microscopy, Staining

Journal: medRxiv

Article Title: Insights into KIF11 pathogenesis in Microcephaly-Lymphedema-Chorioretinopathy syndrome: a lymphatic perspective

doi: 10.1101/2023.11.02.23297090

Figure Lengend Snippet: (a) UMAP visualisation of scRNA-seq data of endothelial cell and related cell populations within wildtype zebrafish at 3 and 4 days post fertilization (dpf). (b) Dotplot of kif11 expression across all reported cell types. Size of circles indicates percentage of cells which express kif11 and the colour range is shaded from high to low expression. (c) UMAP visualisation of kif11 and proliferation markers mki67 and pcna expression, revealing expression of kif11 in proliferating cells. (d) Dotplot showing Spearman correlation of gene expression between kiff11 and mki67 within endothelial cell populations. Size of circle indicates significance value (all correlations p-value < 0.05) and blue to red shading indicates positive or negative correlations, respectively. Value inside dots gives level of correlation. Data from a - d sourced from Grimm and colleagues . (e) Sequencing reads in wildtype and kif11 (8bpdel/8bpdel) zebrafish generated using the CRISPR/Cas9 approach (upper panel). The 8 bp deletion induces a frameshift and subsequent premature stop codon. The predicted Eg5 protein structure of the wildtype and kif11 (8bpdel/8bpdel) zebrafish. The premature stop codon (PSS) induces a truncation within the kinesin motor domain of Eg5 (encoded by kif11 ). (Source: https://www.ebi.ac.uk/interpro/protein/UniProt/F1QK82/ ). (f) Differential inference contrast (DIC) images shows abnormal rostral development in homozygous animals at the 10 somite stage (∼14hpf) and somite defects, neural necrosis and delayed tail extension at 14 somite stage (∼16hpf). Scale = 100µm. (g) Brightfield images at 24 hours post fertilization (hpf) show severe necrosis and global developmental delay within homozygous animals. Confocal images of endothelial cells show impaired intersegmental vessel sprouting and loss of endothelial cell in the head. (h) Brightfield images at 36 hpf demonstrate developmental delay compared to wildtype siblings. Scale =1mm; or =100µm in the dorsal view of heads in right panel. (i) Lateral views of wildtype and kif11 heterozygous trunk vasculature with endothelial nuclei (green) and veins and lymphatics (magenta) labelled. No difference was observed in lymphatic endothelial cell numbers in kif11 heterozygotes compared with controls, n=12 wildtype siblings and n=11 kif11 (+/8bpdel) .

Article Snippet: Eg5 monoclonal antibodies against the N-terminal (CC10014, Cell Applications or NB500-181, Novus Biologicals) were used in the western blots and β-Actin was used as an internal control.

Techniques: Expressing, Sequencing, Generated, CRISPR

Journal: medRxiv

Article Title: Insights into KIF11 pathogenesis in Microcephaly-Lymphedema-Chorioretinopathy syndrome: a lymphatic perspective

doi: 10.1101/2023.11.02.23297090

Figure Lengend Snippet: (a) EG5 ( KIF11 ) protein domain structure indicating the position of the MLC mutations p.L347Efs*8 and p.R387* for which patient-derived lymphoblastoid cells and blood samples were available. (b) qRT-PCR analysis of blood using allele-specific primers (wt for wild type allele and mut for allele containing the mutation) showing strong reduction, but not a complete absence of the mutant mRNA in all analysed samples (F2-I.2, F2-II.1, F1-I.2 and F1-II.1), suggesting nonsense mediated RNA decay. wt KIF11 mRNA levels were decreased by up to 50% in patients compared to the controls. Data represent average relative expression (n=3 experiments) ± SD. (c) Western blot analysis of protein lysates from lymphoblastoid cell lines also indicate approximately 50% reduction in the levels of wt KIF11 protein (119kDa) in the patient samples (F2-I.2, F2-II.1, and F1-II.1) using C-terminal binding EG5 antibody (NB500-181, Novus Biologicals). Experiment shows an average of 2 technical repeats of identical biological materials. Beta-actin was used as a loading control. (d) Western blot analysis with an N-terminal binding anti-EG5 antibody (CC10014, Cell Applications, USA) showed the presence of a truncated protein of the expected size in patient F1-I.2 (arrow). The position of molecular mass markers (in kDa) is indicated on the left of the gel. (e) Top panel: Sanger sequencing of gDNA from proband F5-II.1. PCR product shows a compound peak in exon 20, which is the synonymous KIF11 c.2922G>A; p.(P974=) variant. Lower panel: Sanger sequencing of cDNA from F5-II.1. Black line indicates the boundary between the last base of exon 20 and first base of exon 21. Notice the heteroduplex in exon 20 indicating exon skipping. Analysis of the full trace identified a loss of the last 108 bases in exon 20 (r.2815_2922del) which is predicted to lead to a shorter EG5 protein similar to that of the synonymous variant c.2922G>T as shown by others .

Article Snippet: Eg5 monoclonal antibodies against the N-terminal (CC10014, Cell Applications or NB500-181, Novus Biologicals) were used in the western blots and β-Actin was used as an internal control.

Techniques: Derivative Assay, Quantitative RT-PCR, Mutagenesis, Expressing, Western Blot, Binding Assay, Control, Sequencing, Variant Assay

Journal: Cancer Research

Article Title: Loss of Histone Deacetylase 4 Causes Segregation Defects during Mitosis of p53-Deficient Human Tumor Cells

doi: 10.1158/0008-5472.can-08-2796

Figure Lengend Snippet: Figure 2. Loss of HDAC4 is well tolerated in normal cells. A, expression levels of HDAC4 in human normal tissues show that HDAC4 has a much more tissue-restricted expression, specifically, in muscle and testis, as compared with the ubiquitously expressed HDAC1 and HDAC3. B, parallel transfection of HCT-116 cells and NHDF with HDAC4.13 siRNA led to a cell death phenotype at 72 h posttransfection only in tumor cells, as shown by cell detachment and membrane blebbing (a), despite similar knockdown efficiencies measured at 24 h (b). C, human myelopoietic progenitors transfected with HDAC4 siRNAs; despite down-regulation of HDAC4 expression, no decrease in clonogenicity was observed. GL2 siRNA served as a negative control, and Eg5 siRNA as a positive control.

Article Snippet: HDAC4 and phosphorylated H3 antibodies were from Cell Signaling, a-tubulin antibody was from Sigma, and Eg5 and PARP antibodies were from Novus Biologicals.

Techniques: Expressing, Transfection, Membrane, Knockdown, Negative Control, Positive Control

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: Molecular Biology of the Cell

Article Title: Kinesin molecular motor Eg5 functions during polypeptide synthesis

doi: 10.1091/mbc.E11-03-0211

Figure Lengend Snippet: Eg5 inhibition or microtubule disruption causes a defect in protein synthesis. Quantitation of 35 S Met/Cys incorporation assays in WCLs from RPE1 cells as indicated (A) 4 h after a 130 μM monastrol or DMSO (solvent control) treatment or a 4-h monastrol or DMSO treatment followed by a 4-h release into the same medium without monastrol or DMSO, (B) 24 h before or after Eg5 knockdown (siEg5#1), or (C) a 2-h treatment with 12 μM nocodazole or 0.002 mg/ml Colcemid (microtubule disruptors) or the DMSO solvent control. CHX treatment in A was used as a positive control. In B, a representative immunoblot is shown to demonstrate Eg5 was knocked down 24 h after the addition of siEg5#1 siRNA; actin is used as the loading control. Results are shown as means ± SD and are representative of at least three independent experiments; p values are derived from Student's t test (null hypothesis). (D) Quantification of mitotic indices by DAPI staining after a 130 μM monastrol treatment, 3 μM dimethylenastron, or 1.5 μM S-trityl- l -cysteine for the indicated times or Eg5 knockdown by siEg5#1 or siEg5#2 siRNA for 24 h. DMSO was used as the control for the small-molecule inhibitor treatments, and siControl was used as the control for the knockdown experiments. Asterisks represent the time point or the siRNA at which all translation experiments were completed (except where indicated). There is no significant increase in mitotic indices between the time points labeled with asterisks and controls (0 h or siControl; p > 0.1). Longer treatments of Eg5 inhibition are shown and did lead to a mitotic arrest, but these times were not used in the translation experiments; instead, they are shown to demonstrate that the Eg5 inhibitors were active at the concentration used. In each experiment, a minimum of 300 cells were counted and at least three independent experiments were completed. Results are shown as means ± SD.

Article Snippet: The following antibodies were used in this study: Eg5, rabbit anti-Eg5 (AKIN03; Cytoskeleton, Denver, CO); actin, rabbit anti-actin (AAN01; Cytoskeleton); Eg5, mouse anti-Eg5 (627802; BioLegend, San Diego, CA); RPS5, mouse anti-RPS5 (AB58345; Abcam, Cambridge, MA); RPL10A, mouse anti-RPL10A (Ab55544; Abcam); GAPDH, rabbit anti-GAPDH (14C10) (2118; Cell Signaling, Beverly, MA); calnexin, rabbit anti-calnexin (Stressgen, Enzo Life Sciences, San Diego, CA); caspase-3, rabbit anti–caspase 3 (8G10) (9665; Cell Signaling); FLAG, mouse anti-FLAG (4049; Sigma-Aldrich); HA, mouse anti-HA (1 583 816; Roche, Indianapolis, IN); RPL4, rabbit anti-RPL4 (Proteintech Group, Chicago, IL); and phospho-H3, rabbit anti–phospho-H3 (Upstate, Millipore, Billerica, MA).

Techniques: Inhibition, Disruption, Quantitation Assay, Solvent, Control, Knockdown, Positive Control, Western Blot, Derivative Assay, Staining, Labeling, Concentration Assay

Journal: Molecular Biology of the Cell

Article Title: Kinesin molecular motor Eg5 functions during polypeptide synthesis

doi: 10.1091/mbc.E11-03-0211

Figure Lengend Snippet: Eg5 associates with ribosomes and links ribosomes to microtubules. (A, B) Polysome profiling of mature ribosomes from RPE1 cells. WCLs were placed on a 10–45% continuous sucrose gradient, centrifuged for 2.5 h at 27,000 rpm in a Sorvall AH629 rotor, and fractionated with constant monitoring at an absorbance of 254 nm. (A) Immunoblots are representative of at least six independent experiments; the relative loads for each of the fractions are as follows: 40S and 60S subunits, 50%; 80S ribosomes, 33%; polysomes, 16.7%. Poly, polysomes. (B) Polysome profiling of control RPE1 cells (top) or in the absence of magnesium chloride (MgCl 2 ; bottom). The positions of the 80S ribosomes and polysomes in the control gradient were used to determine the fraction numbers that would be collected as 80S ribosomes or polysomes in the −MgCl 2 , +EDTA sample. The relative loads for each of the fractions are as follows: 40S, 60S subunits, and 80S ribosomes, 50%; polysomes ∼40%. Each of these experiments was completed at least five times. (C) Top, immunoprecipitation of Eg5 in RPE1 cells was conducted and immunoprecipitates were probed for the presence of the 60S ribosomal subunits (rpL10A; left), or for the presence of the 40S ribosomal subunits (rpS5, right). Bottom, reciprocal IP of rpS5 was conducted and immunoprecipitates were probed for the presence of Eg5 (left; separate exposures are shown for input and IP lanes). IP of nonimmune serum was used as a negative control (right); IP of Eg5 was completed in parallel to confirm the association of Eg5 with rpS5. For each sample, 50% of the IP was loaded on the gel; input lane for Eg5 and control IP represents 10% of the total and for rpS5 IP represents 15% of the total. Results shown are representative of at least three independent experiments. −AB, no antibody added; +AB, Eg5/rpS5 antibody added. (D) Sucrose gradient–isolated 80S ribosome fractions from RPE1 WCLs were added to preformed taxol-stabilized microtubules for 45 min, followed by a 30-min centrifugation at 10,000 × g . Immunoblots in the presence of Eg5 (control) are representative of at least five independent experiments and in the absence of Eg5 (siEg5) are representative of at least three independent assays. Antibodies to rpS5 and rpL4 were used to demonstrate the 80S ribosome binding to the pelleted microtubules, and each of these antibodies represents an independent microtubule-binding assay. siEg5#1 siRNA for 24 h was used to knock down Eg5 in this experiment. P, microtubule pellet; S, supernatant. Assays completed in the presence (D; plus microtubules) or (E) in the absence (no MT) of polymerized microtubules.

Article Snippet: The following antibodies were used in this study: Eg5, rabbit anti-Eg5 (AKIN03; Cytoskeleton, Denver, CO); actin, rabbit anti-actin (AAN01; Cytoskeleton); Eg5, mouse anti-Eg5 (627802; BioLegend, San Diego, CA); RPS5, mouse anti-RPS5 (AB58345; Abcam, Cambridge, MA); RPL10A, mouse anti-RPL10A (Ab55544; Abcam); GAPDH, rabbit anti-GAPDH (14C10) (2118; Cell Signaling, Beverly, MA); calnexin, rabbit anti-calnexin (Stressgen, Enzo Life Sciences, San Diego, CA); caspase-3, rabbit anti–caspase 3 (8G10) (9665; Cell Signaling); FLAG, mouse anti-FLAG (4049; Sigma-Aldrich); HA, mouse anti-HA (1 583 816; Roche, Indianapolis, IN); RPL4, rabbit anti-RPL4 (Proteintech Group, Chicago, IL); and phospho-H3, rabbit anti–phospho-H3 (Upstate, Millipore, Billerica, MA).

Techniques: Western Blot, Control, Immunoprecipitation, Negative Control, Isolation, Centrifugation, Binding Assay, Microtubule Binding Assay, Knockdown

Journal: Molecular Biology of the Cell

Article Title: Kinesin molecular motor Eg5 functions during polypeptide synthesis

doi: 10.1091/mbc.E11-03-0211

Figure Lengend Snippet: Eg5 is required for IRES-mediated and 5′ cap-dependent translation initiation. U2OS cells were transfected with Eg5 siRNA (siEg5#1) for a total of 36 h. Eg5 was knocked down in U2OS cells for 12 h prior to cells being transfected with a bicistronic expression plasmid for an additional 24 h. Bar graph represents quantification of immunoblots from five independent experiments with antibodies to HA or FLAG epitopes on the CFP (IRES-mediated initiation, black bars) or YFP (5′ cap-dependent initiation, white bars) marker proteins. Results are represented as means ± SD; p values are derived from Student's t test (null hypothesis).

Article Snippet: The following antibodies were used in this study: Eg5, rabbit anti-Eg5 (AKIN03; Cytoskeleton, Denver, CO); actin, rabbit anti-actin (AAN01; Cytoskeleton); Eg5, mouse anti-Eg5 (627802; BioLegend, San Diego, CA); RPS5, mouse anti-RPS5 (AB58345; Abcam, Cambridge, MA); RPL10A, mouse anti-RPL10A (Ab55544; Abcam); GAPDH, rabbit anti-GAPDH (14C10) (2118; Cell Signaling, Beverly, MA); calnexin, rabbit anti-calnexin (Stressgen, Enzo Life Sciences, San Diego, CA); caspase-3, rabbit anti–caspase 3 (8G10) (9665; Cell Signaling); FLAG, mouse anti-FLAG (4049; Sigma-Aldrich); HA, mouse anti-HA (1 583 816; Roche, Indianapolis, IN); RPL4, rabbit anti-RPL4 (Proteintech Group, Chicago, IL); and phospho-H3, rabbit anti–phospho-H3 (Upstate, Millipore, Billerica, MA).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Marker, Derivative Assay

Journal: Molecular Biology of the Cell

Article Title: Kinesin molecular motor Eg5 functions during polypeptide synthesis

doi: 10.1091/mbc.E11-03-0211

Figure Lengend Snippet: Polysome profiling after loss of Eg5 activity leads to a decrease in the 80S ribosomes and an accumulation of polysomes, indicative of an elongation and/or termination defect. (A) Polysome profiling in RPE1 cells before (left, DMSO + CHX) or after a 4-h 130 μM monastrol treatment (middle, monastrol + CHX) or in the presence of 0.05 mM arsenite (right, arsenite + CHX). CHX was added 10 min prior to cell trypsinization. DMSO + CHX and monastrol + CHX were completed six times, whereas arsenite + CHX was completed twice, all yielding similar results. (B) Same experimental design as in A, except that CHX was omitted. DMSO − CHX and monastrol – CHX were completed four times, whereas arsenite − CHX was completed twice, all yielding similar results. All six of the profiles shown were completed at the same time. Polysome profiling was completed as described in and Materials and Methods . P/M, polysomes/monosomes ratio calculated as described in Materials and Methods .

Article Snippet: The following antibodies were used in this study: Eg5, rabbit anti-Eg5 (AKIN03; Cytoskeleton, Denver, CO); actin, rabbit anti-actin (AAN01; Cytoskeleton); Eg5, mouse anti-Eg5 (627802; BioLegend, San Diego, CA); RPS5, mouse anti-RPS5 (AB58345; Abcam, Cambridge, MA); RPL10A, mouse anti-RPL10A (Ab55544; Abcam); GAPDH, rabbit anti-GAPDH (14C10) (2118; Cell Signaling, Beverly, MA); calnexin, rabbit anti-calnexin (Stressgen, Enzo Life Sciences, San Diego, CA); caspase-3, rabbit anti–caspase 3 (8G10) (9665; Cell Signaling); FLAG, mouse anti-FLAG (4049; Sigma-Aldrich); HA, mouse anti-HA (1 583 816; Roche, Indianapolis, IN); RPL4, rabbit anti-RPL4 (Proteintech Group, Chicago, IL); and phospho-H3, rabbit anti–phospho-H3 (Upstate, Millipore, Billerica, MA).

Techniques: Activity Assay

Journal: Molecular Biology of the Cell

Article Title: Kinesin molecular motor Eg5 functions during polypeptide synthesis

doi: 10.1091/mbc.E11-03-0211

Figure Lengend Snippet: Inhibition of Eg5 slows the rate of ribosome transit. Ribosome half-transit assays before (DMSO) or after (4 h, 130 μM monastrol) Eg5 inhibition in RPE1 cells, as described further in Materials and Methods and Results . Briefly, incorporation of 35 S Met/Cys into total proteins (postmitochondrial supernatants [PMS]; black diamonds) and completed proteins (postribosomal supernatants [PRS]; white diamonds) were graphed over time. The half-transit time was determined by linear regression analysis from the displacement in time between the two x-intercepts. (A) Bar graph represents the average half-transit time of four independent experiments before (DMSO) or after (monastrol) Eg5 inhibition and demonstrated an ∼2.5-fold increase after Eg5 inhibition. (B) A single representative experiment is shown with the calculated half-transit times for this experiment. The DMSO half-transit time was calculated to be 37.4 s (transit time, 1 min, 15 s), whereas after a 4-h monastrol treatment the half-transit time was 143.6 s (transit time, 4 min, 47 s), resulting in an ∼3.8-fold increase for this representative experiment. Results in A are represented as means ± SD; p values are derived from Student's t test (null hypothesis).

Article Snippet: The following antibodies were used in this study: Eg5, rabbit anti-Eg5 (AKIN03; Cytoskeleton, Denver, CO); actin, rabbit anti-actin (AAN01; Cytoskeleton); Eg5, mouse anti-Eg5 (627802; BioLegend, San Diego, CA); RPS5, mouse anti-RPS5 (AB58345; Abcam, Cambridge, MA); RPL10A, mouse anti-RPL10A (Ab55544; Abcam); GAPDH, rabbit anti-GAPDH (14C10) (2118; Cell Signaling, Beverly, MA); calnexin, rabbit anti-calnexin (Stressgen, Enzo Life Sciences, San Diego, CA); caspase-3, rabbit anti–caspase 3 (8G10) (9665; Cell Signaling); FLAG, mouse anti-FLAG (4049; Sigma-Aldrich); HA, mouse anti-HA (1 583 816; Roche, Indianapolis, IN); RPL4, rabbit anti-RPL4 (Proteintech Group, Chicago, IL); and phospho-H3, rabbit anti–phospho-H3 (Upstate, Millipore, Billerica, MA).

Techniques: Inhibition, Derivative Assay

Journal: Molecular Biology of the Cell

Article Title: Kinesin molecular motor Eg5 functions during polypeptide synthesis

doi: 10.1091/mbc.E11-03-0211

Figure Lengend Snippet: Schematic representation of the proposed function of Eg5. Eg5 is suggested to serve as a mobile molecular link between the ribosomes and microtubules to enhance the efficiency of polypeptide synthesis. (A) Eg5 may directly link the 80S ribosomes to microtubules or (B) Eg5 may be indirectly associated with ribosomes via unidentified linker molecules.

Article Snippet: The following antibodies were used in this study: Eg5, rabbit anti-Eg5 (AKIN03; Cytoskeleton, Denver, CO); actin, rabbit anti-actin (AAN01; Cytoskeleton); Eg5, mouse anti-Eg5 (627802; BioLegend, San Diego, CA); RPS5, mouse anti-RPS5 (AB58345; Abcam, Cambridge, MA); RPL10A, mouse anti-RPL10A (Ab55544; Abcam); GAPDH, rabbit anti-GAPDH (14C10) (2118; Cell Signaling, Beverly, MA); calnexin, rabbit anti-calnexin (Stressgen, Enzo Life Sciences, San Diego, CA); caspase-3, rabbit anti–caspase 3 (8G10) (9665; Cell Signaling); FLAG, mouse anti-FLAG (4049; Sigma-Aldrich); HA, mouse anti-HA (1 583 816; Roche, Indianapolis, IN); RPL4, rabbit anti-RPL4 (Proteintech Group, Chicago, IL); and phospho-H3, rabbit anti–phospho-H3 (Upstate, Millipore, Billerica, MA).

Techniques:

Journal: JCI Insight

Article Title: Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

doi: 10.1172/jci.insight.177656

Figure Lengend Snippet: ( A ) EG5 ( KIF11 ) protein domain structure indicating the position of the MLC variants p.L347Efs*8 and p.R387*. ( B ) qRT-PCR analysis of blood using allele-specific primers (mut: patient variant allele) showing strong reduction but not a complete absence of the mutant mRNA in all samples (F2-I.2, F2-II.1, F1-I.2 and F1-II.1), suggesting nonsense-mediated RNA decay. WT KIF11 mRNA levels decreased by up to 50% in patients compared with controls. Data represent average relative expression ( n = 3 experiments) ± SD. ( C ) Western blot analysis of lymphoblastoid cell line lysates indicate approximately 50% reduction in the levels of WT EG5 protein (119 kDa) in the patient samples (F2-I.2, F2-II.1, and F1-II.1) using C-terminal binding EG5 antibody (NB500-181, Novus Biologicals). Average of 2 technical repeats of identical biological materials is shown with β-actin as loading control. ( D ) Western blot analysis with an N-terminal binding anti-EG5 antibody (CC10014, Cell Applications) showed the presence of a truncated protein of the expected size in patient F1-I.2 (arrow). The position of molecular mass markers (in kDa) is indicated on the left of the gel. ( E ) Left panel: Sanger sequencing of gDNA from proband F5-II.1. PCR product shows a compound peak in exon 20, which is the synonymous KIF11 c.2922G>A; p.(P974=) variant. Right panel: Sanger sequencing of cDNA from F5-II.1. Black line indicates the boundary between the last base of exon 20 and first base of exon 21. Notice the heteroduplex in exon 20 indicating exon skipping. Analysis of the full trace identified a loss of the last 108 bases in exon 20 (r.2815_2922del), which is predicted to lead to a shorter EG5 protein similar to that of the synonymous variant c.2922G>T as shown by others .

Article Snippet: Eg5 monoclonal antibodies against the N-terminal (CC10014, Cell Applications or NB500-181, Novus Biologicals) were used in the Western blots, and β-actin was used as an internal control.

Techniques: Quantitative RT-PCR, Variant Assay, Mutagenesis, Expressing, Western Blot, Binding Assay, Control, Sequencing

Journal: JCI Insight

Article Title: Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

doi: 10.1172/jci.insight.177656

Figure Lengend Snippet: Mouse embryonic sections from developmental stages ( A ) E10.5 and ( B ) E12.5 were subjected to immunofluorescence staining. Selected magnified (framed) areas highlight regions containing dermal lymphatics and the thoracic duct, which were imaged for HOECHST (blue; channel 1), Ki67 (green; channel 2), EG5 (red; channel 3), and VEGFR3 (white; channel 4) protein expression. Additionally, whole-mount immunofluorescence staining was performed on ( C ) intestinal and ( D ) ear samples. Selected magnifications highlight regions with lacteals ( C ) and initial lymphatic vessels ( D ), also imaged for Ki67 (green; channel 2), EG5 (red; channel 3), and VEGFR3 (white; channel 4); merged images are also shown. Arrowheads indicate areas with coexpression of EG5 and VEGFR3. Scale bars: 100 μm. Additional labeling was added to facilitate the understanding of the embryonic anatomy: A, artery; LEC, lymphatic endothelial cell; NC, notochord; NT, neural tube; PLV, peripheral lymphatic vessel; PTD, primordial thoracic duct; V, vein.

Article Snippet: Eg5 monoclonal antibodies against the N-terminal (CC10014, Cell Applications or NB500-181, Novus Biologicals) were used in the Western blots, and β-actin was used as an internal control.

Techniques: Immunofluorescence, Staining, Expressing, Labeling