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: 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: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a , Overview of base editing screens to identify drug resistance variants in cancer cell models. b , Base editor screens in HT-29 cells across 11 cancer genes show depletion of gRNAs targeting essential genes demonstrating base editing activity. Unpaired, two-tailed Student’s t -test comparing NT gRNAs ( n = 114) with gRNAs targeting essential gene splice sites ( n = 632) in CBE and ABE screens. Boxplots represent the median, interquartile range (IQR) and whiskers are the lowest and highest values within 1.5 × IQR. c , Comparison of gRNA z -scores from base editor screens in PC9 ( EGFR -mutant, MYC -dependent) and HT-29 ( BRAF -mutant, MYC -dependent) reveals shared and disparate oncogene dependencies. d , Base editing mutagenesis screens of the driving oncogene, BRAF , in HT-29 cells reveal functional protein domains, sites of post-translational modification and driver variants. Data are the average of two independent experiments. See also Extended Data Fig. . Schematic in a created with BioRender.com .

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Activity Assay, Two Tailed Test, Comparison, Mutagenesis, Functional Assay, Modification

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: Replicate correlation for CBE and ABE screens across four cancer cell models; HT-29, H23, PC9 and MHH-ES-1. Pearson correlation coefficient values (r) between independent replicate screens are shown. Low correlation was observed for replicates of PC9 screens with gefitinib, which may relate to a high degree of enrichment of resistant, EGFR T790M base edit harbouring cells.

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques:

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a) Base editor screens in H23, PC9 and MHH-ES-1 cancer cells targeting 11 cancer genes show depletion of gRNAs targeting essential genes demonstrating base editing activity. Unpaired, two-tailed Student’s t-test comparing non-targeting gRNAs ( n = 114) to gRNAs targeting essential gene splice sites ( n = 632) in CBE and ABE screens. For MHH-ES-1, ABE screens are shown (NT; n = 57; essential-targeting, n = 306). Boxplots represent the median and interquartile range (IQR), and whiskers represent the lowest and highest values within 1.5 x the IQR. b) Number of off-target sites plotted against the z-score for base editing gRNAs. A high number of off-targets for a small number of KRAS UTR -targeting gRNAs is associated with severe gRNA depletion. These were filtered out of downstream analysis. c) Our previously reported whole-genome CRISPR-Cas9 KO screen in HT-29 cells in the presence of dabrafenib (0.1 µM) across three time-points. Volcano plot showing EGFR KO as the top sensitising hit. Data are the average of two independent screens and significance was determined with MAGeCK, with a threshold of p-value < 0.05 and FDR < 0.05. d) TCGA oncoprint (pan-cancer cohort, n = 526) of colorectal adenocarcinomas with alterations in KRAS and BRAF . Mutual exclusivity p-value < 0.001 derived from two-sided Fisher exact test, q-value < 0.001 derived from Benjamini-Hochberg FDR correction procedure for multiple hypothesis testing.

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Activity Assay, Two Tailed Test, CRISPR, Derivative Assay

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a , Variants conferring resistance or sensitivity to the MEK inhibitor, trametinib, in HT-29 cells. Comparison of gRNA z -scores for the control treated arm versus plasmid library, and the drug-treated arm versus plasmid library is shown. b , Variants conferring resistance to the combination of BRAF and EGFR inhibitors, dabrafenib and cetuximab, in HT-29 cells. Comparison of gRNA z -scores for the control treated arm versus plasmid library, and the drug-treated arm versus plasmid library is shown. c , Crystal structure of the complex of EGFR and cetuximab (PDB 1yy9 ) , and MEK1 and trametinib (PDB 7jur ) , highlights canonical drug resistance variants discovered in base editor screens predicted to disrupt drug binding. d , Cell growth of base-edited HT-29 cells harboring canonical and drug-addiction drug-resistance variants. Cells were left untreated or treated with trametinib (3 nM) or the combination of dabrafenib (80 nM) and cetuximab (1 µg ml −1 ), and cell proliferation was monitored using an incucyte. Data represent the mean ± s.d. of biological triplicates and are representative of two independent experiments. e , Western blotting of WT HT-29 ABE cells and cells harboring drug-resistance mutations activating the MAPK signaling pathway. Cells were treated with the combination of dabrafenib (80 nM) and cetuximab (1 µg ml −1 ) or DMSO as a control for 24 h before analysis. f , β-galactosidase staining for senescent cells; β-galactosidase positive senescent foci (blue) are indicated with arrows. HT-29 cells were treated with the combination of dabrafenib (80 nM) and cetuximab (1 µg ml −1 ) or DMSO as a control for 48 h before analysis. Representative images are shown for the drug addiction variant MAP2K1 Y130C. Scale bar, 500 µm. Predicted amino acid editing consequences are labeled for drug resistance screens and genotyped edits are shown in d , e and f . Data are the average of two independent experiments performed on separate days, or representative of two independent experiments ( e and f ). See also Extended Data Figs. and .

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Comparison, Control, Plasmid Preparation, Binding Assay, Western Blot, Staining, Variant Assay, Labeling

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a , Drug resistance variants to the EGFR inhibitor gefitinib, profiled with CBE and ABE base editors in PC9 lung cancer cells. Comparison of gRNA z -scores for the control treated arm versus plasmid library, and the drug-treated arm versus plasmid library is shown. b , Drug resistance variants to the EGFR inhibitor, osimertinib, profiled with CBE and ABE base editors in PC9 lung cancer cells. Comparison of gRNA z -scores for the control treated arm versus plasmid library, and the drug-treated arm versus plasmid library is shown. Data represent the average of two independent screens performed on separate days. c , Prime editing mutagenesis screens of EGFR in the presence and absence of osimertinib. PC9 ∆ MLH1 cells were prime edited for 7 days with doxycycline (1 µg ml −1 ) before growth for 10 days in DMSO (control) or osimertinib (75 nM). Data represent the z -score for each pegRNA derived from the average of two independent screens performed on separate days. Samples were compared with the plasmid library. d , Competition flow cytometry assays in PC9 ∆ MLH1 cells comparing the growth of NT gRNA GFP cells with epegRNA BFP cells harboring different EGFR variants in the presence and absence of osimertinib (75 nM) for 5 days. Data are normalized to day 0 ratios and represent the mean ± s.d. of biological triplicates. Unpaired, two-tailed Student’s t -test comparing with the EGFR C797C synonymous variant control; * P = 0.0003, ** P = 0.0002, *** p < 0.0001. Predicted amino acid editing consequences are labeled for drug resistance variant screens. See also Extended Data Fig. .

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Comparison, Control, Plasmid Preparation, Mutagenesis, Derivative Assay, Flow Cytometry, Two Tailed Test, Variant Assay, Labeling

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a) Western blot for MLH1 verifies KO of MLH1 in PC9 cells. PC9 cells were transfected with a Cas9-GFP plasmid encoding an MLH1 targeting gRNA. FACS of GFP positive single cells gave clonal populations, or a pooled population (“pool”). Cells were expanded before analysis by Western blotting. Actin serves as a loading control. Data are representative of two independent experiments. b) Sanger sequencing of prime editing of EGFR C797S in PC9 cells. PC9-PE MLH1 KO (clone 1 from above), or MLH1 WT PC9-PE cells were infected with a pegRNA encoding the C797S edit, puromycin selected and prime editing was initiated with the addition of doxycycline for 5 days. Control (untreated) or osimertinib selected cells (5 nM) are shown. The EGFR C797 locus was PCR amplified and then analysed with Sanger sequencing. c) Replicate correlation between pegRNA z-scores from EGFR prime editing mutagenesis screens performed in PC9 MLH1 KO PE2 cells. Data are from two independent screens performed on different days. Labelled are predicted mutations in EGFR installed by the pegRNAs. Pearson correlation coefficient values (r) between independent replicate screens are shown. pegRNA, prime editing gRNA.

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Sequencing, Infection, Amplification, Mutagenesis

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a) Drug titration experiments in PC9 CBE and ABE cells using Cell-titre Glo to measure cell proliferation in the presence of EGFR inhibitors (cetuximab, erlotinib, lapatinib), or chemotherapy agents (cisplatin, paclitaxel). Data represent the mean ± SD of two independent experiments performed on separate days, each in biological triplicate. b) Sanger sequencing of DNA from WT or base edited PC9 cells harbouring the EGFR-inhibitor sensitising splice variant. CBE editing and ABE editing of a known (GT) splice donor is shown. The position of each gRNA is indicated. c) Sanger sequencing cDNA from WT or base edited PC9 cells harbouring the EGFR-inhibitor sensitising splice variant. WT cells display exon-exon splicing as expected, whereas mutant cells display intron retention by utilising an alternative splice donor in the downstream intron. d) Gating strategy for flow cytometry analysis of EGFR expression on PC9 cells (FITC). Gating was performed on cells, singlets, viable cells, BFP+ cells (gRNA expression).

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Titration, Sequencing, Variant Assay, Mutagenesis, Flow Cytometry, Expressing

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a , Increased gefitinib and osimertinib sensitivity in PC9 cells with EGFR C-terminal truncating mutations. Data represent the mean ± s.e.m. of two independent experiments, each performed in biological triplicate. Two-way ANOVA (analysis of variance) comparing with parental (Par.) response; *** P < 0.0001. CTG; CellTiter-Glo. b , A drug-sensitizing base edit in EGFR causes loss of a splice donor site. The EGFR RNA splice variants are shown by migration of PCR products from cDNA. The larger PCR product in the mutant samples is due to retention of a short region of a downstream intronic sequence after exon 27, where an alternative splice donor is used. EGFR protein after residue 1,091 is not translated due to a frameshift leading to a stop codon. c , Western blotting of drug-sensitizing mutants reveals a C-terminal truncation in EGFR and confirms drug sensitization. PC9 CBE or ABE control cells (NT gRNA) or cells mutant for EGFR were treated with gefitinib (gefit.), osimertinib (osim.) or DMSO vehicle control (ø) for 24 h before analysis. Data are representative of two independent experiments. d , Flow cytometry analysis of EGFR protein surface expression in PC9 cells with WT EGFR or base-edited EGFR. Data are represented as a histogram or quantified as EGFR-FITC mean fluorescence intensity (MFI), and represent the mean of three independent experiments ± s.d. Unpaired, two-tailed Student’s t -test; *** P = 0.0004, ** P = 0.0096, * P = 0.0217. See also Extended Data Fig. .

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Migration, Mutagenesis, Sequencing, Residue, Western Blot, Control, Flow Cytometry, Expressing, Fluorescence, Two Tailed Test

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: Figure 1. Representative images of immunostained tissue microarrays from the colon adenocarcinoma samples with immunoreactivity scores for NEK9, EG5, and acetyl-α-tubulin (magnification, × 200). NEK9, EG5, and acetyl-α-tubulin were thereby grouped into low- (score 0–1) or high- (score 2–3) expression groups.

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 -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: Figure 2. 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 -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: Figure 3. Concordant expression of the NEK9–G5 axis during the G2/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 (pNEK9T210), NEK9, EG5, acetyl- tubulin, phosphorylated AKT (pAKTS473) and cyclin B1 at the indicated times of nocodazole–induced G2/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 -catenin (1:200, clone 14, Cell Marque).

Techniques: Expressing, Blocking Assay, Western Blot