paurka  (Novus Biologicals)

Journal: bioRxiv

Article Title: Aurora kinase A enables collective invasion and metastasis by endowing a leader cell phenotype and stabilizing Eplin-mediated cohesion with follower cells

doi: 10.64898/2026.03.31.715024

Figure Lengend Snippet: a. Uniform manifold approximation and projection (UMAP) including all malignant cells from breast, liver, and axilla biopsies. G2/M scores and AURKA expression are overlayed onto the UMAP. Data sourced from . b. Hallmark Gene Sets plot of fold change differences for Axilla versus Breast and Liver versus Breast highlighting gene sets that are both significantly different and have an absolute fold change value of 5 or more are highlighted with sets enriched in breast (red) or enriched at metastatic sites (blue). Data sourced from . c. Heatmap of correlation between AURKA expression against gene sets/pathways annotated with “metastasis” or “invasion” in Molecular Signatures Database of GSEA. * indicate FDR-adjusted p-value. Data sourced from .

Article Snippet: The following primary antibodies were used: Alexa-647-beta-tubulin (TUBB) (ThermoFisher, MA5-16308-A647, 1:100), K14 (Abcam,181595, 1:1000), cytokeratin 14 (Abcam, ab181595, 1:1000), GFP (Abcam, ab183734, 1:200), phosphorylated AURKA (pAURKA) (Cell Signalling, 3079, 1:1000), AURKA (Abcam, ab13824, 1:200 or Cell Signaling, 3092S, 1:400), E-cadherin (Cell Signaling, 3195S, 1:300 or Cell Signalling, 14472S, 1:300), EPLIN (Cell Signalling, 16639-1-AP, 1:100 or Cell Signalling, sc-136399, 1:100) and gamma-tubulin (TUBG1) (Sigma, T6557, 1:1000).

Techniques: Expressing

Journal: bioRxiv

Article Title: Aurora kinase A enables collective invasion and metastasis by endowing a leader cell phenotype and stabilizing Eplin-mediated cohesion with follower cells

doi: 10.64898/2026.03.31.715024

Figure Lengend Snippet: a. Western blot analysis of parental MCF10A RFP-TUBA1B (WT), GFP-luc and GFP-AURKA/GFP-luc clones, stained for AURKA and GAPDH. b. MCF10A cells were either implanted onto the CAM as a onplant or injected into the amniotic cavity. c. Representative brightfield images over 6 days of growth for onplants (1 x 10 6 cells per onplant) following their engraftment onto embryonic day (ED) 13 chick embryos. d. H&E images (inset) and immunofluorescence (IF) analysis of FFPE CAM isolated at day 6. The presence of human cells was confirmed through IF detection of GFP and a human-specific nucleolin. Human cells were also stained for their expression of K14 and E-cadherin, or vimentin and E-cadherin. Human cells were found as acini structures in both (1) GFP-luc and (2) GFP-AURKA/GFP-luc injected onplants. Human cells were found as cell strand structures in (3) GFP-AURKA/GFP-luc injected onplants. The tips of cell strand structures (boxes) showed positive staining for K14/E-cadherin/vimentin suggestive of a hybrid EMT phenotype. e. Representative bioluminescence imaging, through IVIS, of chick embryos collected six days after injection with either GFP-luc or GFP-AURKA/GFP-luc cells. f. Quantitation of background subtracted whole chicken embryo bioluminescence imaging of embryos injected with either GFP-luc cells or GFP-AURKA/GFP-luc cells. P values calculated by using the Mann Whitney U-test. g. Summary of injection experiments for GFP-luc cells (n=16) or GFP-AURKA/GFP-luc (n=33). h. Immunofluorescence analysis of luciferase-positive chick embryo tissues, previously injected with GFP-AURKA/GFP-luc cells, stained with GFP or E-cadherin.

Article Snippet: The following primary antibodies were used: Alexa-647-beta-tubulin (TUBB) (ThermoFisher, MA5-16308-A647, 1:100), K14 (Abcam,181595, 1:1000), cytokeratin 14 (Abcam, ab181595, 1:1000), GFP (Abcam, ab183734, 1:200), phosphorylated AURKA (pAURKA) (Cell Signalling, 3079, 1:1000), AURKA (Abcam, ab13824, 1:200 or Cell Signaling, 3092S, 1:400), E-cadherin (Cell Signaling, 3195S, 1:300 or Cell Signalling, 14472S, 1:300), EPLIN (Cell Signalling, 16639-1-AP, 1:100 or Cell Signalling, sc-136399, 1:100) and gamma-tubulin (TUBG1) (Sigma, T6557, 1:1000).

Techniques: Western Blot, Clone Assay, Staining, Injection, Immunofluorescence, Isolation, Expressing, Imaging, Quantitation Assay, MANN-WHITNEY, Luciferase

Journal: bioRxiv

Article Title: Aurora kinase A enables collective invasion and metastasis by endowing a leader cell phenotype and stabilizing Eplin-mediated cohesion with follower cells

doi: 10.64898/2026.03.31.715024

Figure Lengend Snippet: a. Images from live-cell imaging of MCF10A RFP-TUBA1B scratch wound assay. Leader cells were identified at 6 hours by presence of lamellipodium and the dynamics of microtubule organization were retrospectively followed in leader cells and follower cells (left). Composite, overlay images of 20 leader or follower cells were generated by ImageJ (right). b. Representative images of a scratch wound assay of MCF10A RFP-TUBA1B cells. Cells were fixed six hours after wounding and stained with AURKA and K14. White arrowhead points to the leader cell. c-d. The proportion of leader and follower cells that are (c) K14-positive or (d) AURKA-positive at 3-hours or 6-hours post-wounding. Data are represented as mean ± SEM, n= 3 experiments. e-g. Scratch wound assay for parental (40 wounds), GFP-CTRL (#1=44, #2=42, #3=31 wounds), and GFP-AURKA (#1=43, #2=47, #3=27 wounds) cells imaged every 30 minutes over the course of 24 hours measuring (f) the percentage of wound closure or (g) the number of migrating cell groups (yellow stars) were quantified. Data are represented as (f) mean ± SEM or (g) mean ± SD, n= 3 experiments, P-value from multiple comparisons two-way ANOVA method t-test. h. Depiction of scratch wound assay with co-culturing of GFP-CTRL or GFP-AURKA with parental cells at a ratio of 1:10, growth for 48 hours until confluent, serum-starvation, and wounding. i-j. Cells were fixed at 6 hours post-scratch and stained with GFP. GFP-positive cells were frequently paired, indicating cell division that occurred post-seeding. Leader cells indicated by an arrowhead. (j) Probability of GFP-CTRL or GFP-AURKA cells becoming leader cells indicates a bias for GFP-AURKA cells. Data are represented as mean ± SEM, n= 2 experiments, P-value from Student’s t-test.

Article Snippet: The following primary antibodies were used: Alexa-647-beta-tubulin (TUBB) (ThermoFisher, MA5-16308-A647, 1:100), K14 (Abcam,181595, 1:1000), cytokeratin 14 (Abcam, ab181595, 1:1000), GFP (Abcam, ab183734, 1:200), phosphorylated AURKA (pAURKA) (Cell Signalling, 3079, 1:1000), AURKA (Abcam, ab13824, 1:200 or Cell Signaling, 3092S, 1:400), E-cadherin (Cell Signaling, 3195S, 1:300 or Cell Signalling, 14472S, 1:300), EPLIN (Cell Signalling, 16639-1-AP, 1:100 or Cell Signalling, sc-136399, 1:100) and gamma-tubulin (TUBG1) (Sigma, T6557, 1:1000).

Techniques: Live Cell Imaging, Scratch Wound Assay Assay, Generated, Staining

Journal: bioRxiv

Article Title: Aurora kinase A enables collective invasion and metastasis by endowing a leader cell phenotype and stabilizing Eplin-mediated cohesion with follower cells

doi: 10.64898/2026.03.31.715024

Figure Lengend Snippet: a. Live-cell imaging of scratch wound assays treated with DMSO or MLN8237 (1μM, 2.5μM, 5μM). Images were taken every 15 minutes over the course of 24 hours. At 12 hours, the wound is outlined by a yellow line and migrating cell clusters are marked by an asterisk (*). Scattered cells are highlighted with a yellow contour. b-d. Live-cell images were used to quantify (b) the wound closure speed (c) migrating cell clusters and (d) scattered cells for cells treated with DMSO (36 wounds) or MLN8237 at a concentration of 1μM (27 wounds), 2.5μM (35 wounds) and 5μM (36 wounds). Data are represented as mean ± SD, n = 3 independent experiments, P-value from multiple comparisons two-way ANOVA method t-test. e. The migration of leader cells with or without the addition of MLN8237 (5 μM) at 0 hours post-scratch were tracked. Leader cells were identified retrospectively at 6 hours using 10x objective images. f. Velocity of leader cells with or without the addition of MLN8237 (5 μM) were determined. g. Schematic of scratch wound assay with or without treatment of MLN8237 or MG132. After making the scratch, media with DMSO was added and cells were allowed to migrate. At 3 hours post-scratch, media with or without treatment of MLN8237 (5μM) or MG132 (1 μM) was added. h. Live-cell imaging was used to observe scratch wound assays treated with or without MLN8237 (5 μM) for 6 hours post-scratch. Leader cells are outlined in green, and two follower cells are outlined in orange. White arrows represent migration vector of the leader cell. i. Quantification of the difference between the migration vector from 0-3 hours and 3-6 hours in response to MLN8237 or MG132. Data are represented as mean ± SEM, n = 3 independent experiments, P-value from multiple comparisons two-way ANOVA method t-test. j. IF analysis of AURKA and E-cadherin expression in migrating MCF10A RFP-TUBA1B cells. The migrating cell cluster have a leader cell (white arrowhead) with high AURKA expression and followers that are positive for E-cadherin. Intercellular bridges between cells at the edge of the wound are also E-cadherin positive. k. Scratch wound assays treated with DMSO or MLN8237 ( 5μM) and stained for AURKA and E-cadherin. l. Line scan analysis performed on immunofluorescence images and quantified for scratch wounds treated with DMSO, MG132, MLN8237 or both MLN8237 + MG132.

Article Snippet: The following primary antibodies were used: Alexa-647-beta-tubulin (TUBB) (ThermoFisher, MA5-16308-A647, 1:100), K14 (Abcam,181595, 1:1000), cytokeratin 14 (Abcam, ab181595, 1:1000), GFP (Abcam, ab183734, 1:200), phosphorylated AURKA (pAURKA) (Cell Signalling, 3079, 1:1000), AURKA (Abcam, ab13824, 1:200 or Cell Signaling, 3092S, 1:400), E-cadherin (Cell Signaling, 3195S, 1:300 or Cell Signalling, 14472S, 1:300), EPLIN (Cell Signalling, 16639-1-AP, 1:100 or Cell Signalling, sc-136399, 1:100) and gamma-tubulin (TUBG1) (Sigma, T6557, 1:1000).

Techniques: Live Cell Imaging, Concentration Assay, Migration, Scratch Wound Assay Assay, Plasmid Preparation, Expressing, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Aurora kinase A enables collective invasion and metastasis by endowing a leader cell phenotype and stabilizing Eplin-mediated cohesion with follower cells

doi: 10.64898/2026.03.31.715024

Figure Lengend Snippet: a. Live-cell imaging of scratch wound treated with DMSO or MLN8237 using SPY555-tubulin and SPY650-FastAct dye to stain microtubules and actin. White arrowheads point to leader cells. Black arrowheads point to altered and aligned microtubule and actin cytoskeleton in MLN8237-treated cells. Dotted region represents leader cell mask used to center inverted images. Temporal color-coded image of microtubules and actin within a leader cell is shown for 2-hour imaging. b. Venn diagram showing 17 overlapping genes between published datasets of the AURKA interactome and the AURKA phosphoproteome . c. Immunoprecipitation of MCF10A migration-enriched cells using EPLIN (left) or AURKA (right) antibodies, then blotted with EPLIN antibody. d. Super-resolution microscopy images of leader cell with follower cells connected with EPLIN and E-cadherin positive intercellular bridges (zoom image). e. Super-resolution microscopy images EPLIN and E-cadherin cell contacts and relocalization of EPLIN to lamellipodia following exposure to MLN8237. f. DMSO (18 cells) or MLN8237 (23 cells) treated scratch wounds were analyzed for EPLIN intensity at the lamellipodia edge/lamella at 6 hours post-scratch. P values were calculated by Student’s t-test.

Article Snippet: The following primary antibodies were used: Alexa-647-beta-tubulin (TUBB) (ThermoFisher, MA5-16308-A647, 1:100), K14 (Abcam,181595, 1:1000), cytokeratin 14 (Abcam, ab181595, 1:1000), GFP (Abcam, ab183734, 1:200), phosphorylated AURKA (pAURKA) (Cell Signalling, 3079, 1:1000), AURKA (Abcam, ab13824, 1:200 or Cell Signaling, 3092S, 1:400), E-cadherin (Cell Signaling, 3195S, 1:300 or Cell Signalling, 14472S, 1:300), EPLIN (Cell Signalling, 16639-1-AP, 1:100 or Cell Signalling, sc-136399, 1:100) and gamma-tubulin (TUBG1) (Sigma, T6557, 1:1000).

Techniques: Live Cell Imaging, Staining, Imaging, Immunoprecipitation, Migration, Super-Resolution Microscopy