Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 1. Emerin protein expression in MCF7, scrambled shRNA, and MCF7 emerin shRNA-transfected cell lines. (A) Representative western blot and (B) quantification of MCF7, scrambled shRNA, and three emerin shRNA cell lines normalized to γ-tubulin. *P = 0.0108, **P = 0.0061, N = 10–13, One-way ANOVA followed by Dunnett’s multiple comparison.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, shRNA, Transfection, Western Blot, Comparison

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 2. Reducing emerin in MCF7 cells decreased nuclear area. (A) Representative DAPI images of MCF7, control shRNA, emerin shRNA, and scrambled shRNA cell lines, which were used to measure nuclear area. (B) Violin plot of nuclear area of MCF7, control shRNA, emerin shRNA, and scrambled shRNA MCF7 cells (N > 50 nuclei). The mean is depicted as the dark dashed line and the thin dashed lines represent the first and third quartiles. *P < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparison.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Control, shRNA, Comparison

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 3. Emerin reduction decreases nuclear volume and increases nuclear bulges and indentations in MCF7 cells. (A) Representative confocal images of DAPI-stained nuclei from MCF7, control shRNA, and emerin shRNA lines. (B) Violin plots of nuclear volumes (N > 15 nuclei for each) of MCF7, control shRNA, and emerin shRNA MCF7 cell lines. The mean is depicted as the dark dashed line and the thin dashed lines represent the first and third quartiles. *P < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparison test. (C) 3-D rendering of z-stacks from representative nuclei in B showing the concavity and convexity of the nuclei. Red– orange indicates a concave surface and green indicates a convex surface. (D) The proportion of deformed nuclei in MCF7, con shRNA, and emerin shRNA MCF7 cells.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Staining, Control, shRNA, Comparison

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 4. Reducing emerin in MCF7 cells increases impeded migration. (A) Violin plot of the number of cells migrating through 8 µm trans-well pores is shown for MCF7, control shRNA, emerin shRNA, and scrambled shRNA cell lines (n = 5 fields). The mean is depicted as the dark dashed line and the thin dashed lines represent the first and third quartiles. *P < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparison. (B) Representative DAPI images of the cells that successfully migrated in the trans-well assays in A. (C) Scratch- wound healing assay. MCF7, control shRNA, emerin shRNA, and scrambled shRNA MCF7 cell lines were plated, scratched with a pipette tip, and their migration into the wound area was monitored over three days. Representative phase images are shown. (D) The rate of scratch wound healing, which refers to the ability of cells to migrate into the wound area is shown with standard error of the mean. Two-way ANOVA did not identify significance.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Migration, Control, shRNA, Comparison, Wound Healing Assay, Transferring

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 5. Reducing emerin in MDA-231 lines fails to affect nuclear size. (A) Representative western blot of emerin and γ-tubulin (loading control) in MDA-231, control shRNA, and emerin shRNA cell lines and (B) quantitation of the western blots, N = 5. P = 0.0018. (C) Representative images of nuclei in MDA-231, control shRNA, and emerin shRNA stable cell lines that were used to measure nuclear area. (D) A violin plot of nuclear area of MDA-231, control shRNA, and emerin shRNA MDA-231 cell lines. The mean is depicted as the dark dashed line and the thin dashed lines represent the first and third quartiles *P < 0.0006, N > 50 nuclei; one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Western Blot, Control, shRNA, Quantitation Assay, Stable Transfection, Comparison

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 6. Reducing emerin in MDA-231 cells fails to decrease nuclear volume but increases nuclear deformations. (A) Representative confocal images of DAPI-stained nuclei from MDA-231, control shRNA, and emerin shRNA MDA-231 cell lines. (B) Violin plots of nuclear volumes (N > 15 nuclei for each) of MDA-231, control shRNA, and emerin shRNA cell lines. The mean is depicted as the dark dashed line and the thin dashed lines represent the first and third quartiles. No significant differences were seen using one-way ANOVA. (C) Representative images of convexity and concavity of nuclei in the respective cell lines. Red–orange indicates concave surface and green indicates a convex surface. (D) Fraction of nuclei with and without deformities for MDA-231, control shRNA, and emerin shRNA cell lines.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Staining, Control, shRNA

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 7. Reducing emerin in MDA-231 cells increases their impeded migration. (A) A violin plot of the number of cells migrating through 8 µm trans-well pores is shown for MDA-231, control shRNA, emerin shRNA, and scrambled shRNA MDA-231 cell lines (N = 5 fields). The mean is depicted as the dark dashed line and the thin dashed lines represent the first and third quartiles. *P < 0.0037 compared to control shRNA, one-way ANOVA followed by Dunnett’s multiple comparison; 3 biological replicates were used for each cell line. (B) Representative DAPI images of the cells that successfully migrated in the trans-well assays in A. (C) Scratch- wound healing assay. MDA-231, control shRNA, emerin shRNA, and scrambled shRNA MDA-231 cell lines were plated, scratched with a pipette tip, and migration into the wound area was monitored every 2 h for 8 h. Representative phase images are shown. (D) The rate of scratch wound healing, which refers to the ability of cells to migrate into the wound area, is shown with SEM. Two-way ANOVA was used, and no significant differences were seen.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Migration, Control, shRNA, Comparison, Wound Healing Assay, Transferring

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 8. Reduction of emerin increases cell proliferation in MCF7 and MDA-231 cells. (A) Growth curves of MCF7, emerin shRNA MCF7, and control shRNA MCF7 cells, as shown by measuring metabolic activity with Presto Blue Cell Viability Reagent (Life Technologies, cat#: A13261) per manufacturer’s instructions. Mean data plotted with SEM; N = 3 biological replicates. * indicates a difference between MCF7 + emerin shRNA and MCF7 cells (*P < 0.05)), as determined by two-way ANOVA and Dunnett’s test. (B) Growth curves of MDA-231, emerin shRNA MDA-231, and control shRNA MDA-231 cell lines as determined using Presto Blue. Mean data plotted with SEM; N = 3 biological replicates. * indicates a significant difference between MDA-231 + emerin shRNA and MDA-231 cells (*P < 0.05), as determined by two-way ANOVA and Dunnett’s test.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: shRNA, Control, Activity Assay

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 10. Graphical hypothesis demonstrating the effect of emerin levels on the progression of metastatic disease.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques:

Journal: Molecular cancer research : MCR

Article Title: Defects in Emerin-nucleoskeleton binding disrupt nuclear structure and promote breast cancer cell motility and metastasis

doi: 10.1158/1541-7786.MCR-20-0413

Figure Lengend Snippet: (A) Western Blot analysis of emerin in primary mammary epithelial cells, MCF10A cells, MDA-231 cells and MDA-157 cells. (B) Western Blot quantification of emerin expression for each cell line. Emerin expression was normalized to tubulin and primary mammary epithelial cells. Error bars represent standard error. (n=3) ****p-value < 0.0001, unpaired t-test. (C) Nuclear area for MCF10A (n=116, blue), MDA-231 (n=237, green) and MDA-157 (n=342, red) cells. Error bars represent standard error. ****p-value < 0.0001, unpaired t-test (D) Representative DAPI (blue) images of MCF10A, MDA-231, and MDA-157 cells. Scale bars: 100 μm. (E) Nuclear circularity for MCF10A (n=75, blue), MDA-231 (n=301, green), and MDA-157 (n=237, red) cells. Error bars represent standard error. ****p-value < 0.0001, unpaired t-test

Article Snippet: Primary antibodies used for blotting included rabbit emerin polyclonal antibody (1:5,000 dilution; Proteintech, Rosemont, IL, cat#: 10351-I-AP), mouse gamma-tubulin monoclonal antibody (1:10,000 dilution; Sigma-Aldrich, cat#: T6557, RRID:AB_532292), mouse actin monoclonal antibody (1:2,500 dilution; Sigma-Aldrich, cat#: A5441, RRID:AB_476744), rabbit H3K9me2 polyclonal antibody (1:5,000 dilution; Active Motif, cat#: 39239, RRID:AB_2793199), rabbit H3K9me3 polyclonal antibody (1:10,000 dilution; Abcam, Cambridge, UK, cat#: ab8898, RRID:AB_306848), and rabbit H3K27me3 polyclonal antibody (1:10,000 dilution; MilliporeSigma, cat#: 07–449, RRID:AB_310624).

Techniques: Western Blot, Expressing

Journal: Molecular cancer research : MCR

Article Title: Defects in Emerin-nucleoskeleton binding disrupt nuclear structure and promote breast cancer cell motility and metastasis

doi: 10.1158/1541-7786.MCR-20-0413

Figure Lengend Snippet: (A) Fluorescence intensity of the primary tumor was measured weekly for eight weeks. (B) Primary tumor size after excision at week eight. n=38 for iRFP713, n=30 for GFP-emerin, n=28 for each GFP-emerin mutant. Error bars represent standard error. *p-value = 0.0004, **p-value = 0.0050, ***p-value = 0.0006, ****p-value= 0.0266, one-way ANOVA. C) Volume of excised tumors at eight weeks post-injection with MDA-231 cells expressing iRFP713 (n=38), and GFP-emerin (n=30), GFP-S54F (n=28), GFP-M196 (n=28), GFP-M45A (n=28), or GFP-M151 (n=28) cells. Error bars represent standard error. *p-value = 0.0005, **p-value = 0.0015, ***p-value = 0.0239, one-way ANOVA (D) Representative fluorescent images of excised primary tumors at eight weeks post-injection. Heat-map shows iRFP713 fluorescence intensity. Scale bars: 1 cm (E) Fluorescence intensity of lung metastasis at eight weeks post-injection. Data was normalized to control lungs with no metastasis. n=38 for iRFP713 alone, n=30 for GFP-emerin, n=28 for each GFP-emerin mutant. Error bars represent standard error. *p-value = 0.0027, **p-value = 0.0040, ***p-value = 0.0034, ****p-value = 0.0156, *****p-value = 0.0107, one-way ANOVA. (F) Lung metastasis fluorescence intensity from mice whose primary tumors were greater than 50,000 FU is shown. Data was normalized to control lungs with no metastasis. n=28 for iRFP713, n=12 for GFP-emerin, n=20 for GFP-S54F, n=16 for GFP-M196, n=24 for GFP-M45A, n=28 for GFP-M151. Error bars represent standard error. *p-value = 0.0023, **p-value = 0.0035, ***p-value = 0.0331, ****p-value = 0.0136, *****p-value = 0.0006, one-way ANOVA. (G) Representative fluorescent images of excised lungs at eight weeks post-injection. Heat-map shows iRFP713 fluorescent intensity.

Article Snippet: Primary antibodies used for blotting included rabbit emerin polyclonal antibody (1:5,000 dilution; Proteintech, Rosemont, IL, cat#: 10351-I-AP), mouse gamma-tubulin monoclonal antibody (1:10,000 dilution; Sigma-Aldrich, cat#: T6557, RRID:AB_532292), mouse actin monoclonal antibody (1:2,500 dilution; Sigma-Aldrich, cat#: A5441, RRID:AB_476744), rabbit H3K9me2 polyclonal antibody (1:5,000 dilution; Active Motif, cat#: 39239, RRID:AB_2793199), rabbit H3K9me3 polyclonal antibody (1:10,000 dilution; Abcam, Cambridge, UK, cat#: ab8898, RRID:AB_306848), and rabbit H3K27me3 polyclonal antibody (1:10,000 dilution; MilliporeSigma, cat#: 07–449, RRID:AB_310624).

Techniques: Fluorescence, Mutagenesis, Injection, Expressing, Control

Journal: Molecular cancer research : MCR

Article Title: Defects in Emerin-nucleoskeleton binding disrupt nuclear structure and promote breast cancer cell motility and metastasis

doi: 10.1158/1541-7786.MCR-20-0413

Figure Lengend Snippet: (A) Nuclear area of MDA-231 cells (n=237, black), MDA-231 cells with no plasmid, but electroporated (electroporation control; n=195, red), and MDA-231 cells expressing GFP (n=53, orange) or GFP-emerin (n=157, blue). Error bars represent standard error. ****p-value < 0.0001, unpaired t-test (B) Representative DAPI (blue) and GFP-emerin (green) images of MDA-231 cells. Scale bar: 50 μm. (C) Nuclear area of MCF10A cells (n=65, black), MCF10A cells with no plasmid, but electroporated (electroporation control; n=45, red), and MCF10A cells expressing GFP (n=90, orange) or GFP-emerin (n=76, blue). Error bars represent standard error. All comparisons had p-values > 0.05. (D) Representative DAPI (blue) and GFP-emerin (green) images of MCF10A cells. Scale bar: 50 μm.

Article Snippet: Primary antibodies used for blotting included rabbit emerin polyclonal antibody (1:5,000 dilution; Proteintech, Rosemont, IL, cat#: 10351-I-AP), mouse gamma-tubulin monoclonal antibody (1:10,000 dilution; Sigma-Aldrich, cat#: T6557, RRID:AB_532292), mouse actin monoclonal antibody (1:2,500 dilution; Sigma-Aldrich, cat#: A5441, RRID:AB_476744), rabbit H3K9me2 polyclonal antibody (1:5,000 dilution; Active Motif, cat#: 39239, RRID:AB_2793199), rabbit H3K9me3 polyclonal antibody (1:10,000 dilution; Abcam, Cambridge, UK, cat#: ab8898, RRID:AB_306848), and rabbit H3K27me3 polyclonal antibody (1:10,000 dilution; MilliporeSigma, cat#: 07–449, RRID:AB_310624).

Techniques: Plasmid Preparation, Electroporation, Control, Expressing

Journal: Molecular cancer research : MCR

Article Title: Defects in Emerin-nucleoskeleton binding disrupt nuclear structure and promote breast cancer cell motility and metastasis

doi: 10.1158/1541-7786.MCR-20-0413

Figure Lengend Snippet: (A) Western blot of MCF10A cells, MDA-231 cells and MDA-231 cells stably expressing GFP-emerin or each GFP-emerin mutant. (B) Endogenous emerin, GFP-emerin and total emerin protein expression was normalized to gamma-tubulin and wildtype emerin in MDA-231 cells. Error bars represent standard deviation. (C) Panel showing known emerin-binding proteins and their disruption by the indicated emerin mutant protein. +, indicates emerin mutant binds to specific binding partner; -, indicates emerin mutant disrupts binding to the protein [58]. Disruptions are highlighted in yellow for clarity. (D) Nuclear area of MDA-231 cells (n=237, black), MDA-231 cells with no plasmid, but electroporated (electroporation control; n=195, black), and MDA-231 cells expressing GFP (n=53, orange), GFP-emerin (n=157, blue), GFP-M45A (n=142, green), GFP-S54F (n=145, purple), GFP-M151 (n=155, yellow), GFP-M24 (n=158, pink) or GFP-M196 (n=149, cyan). Error bars represent standard error. ****p-value < 0.0001, one-way ANOVA. (E) Representative DAPI (blue) and GFP (green) images for GFP-emerin and each GFP-emerin mutant analyzed in D. Scale bars: 50 μm. (F) Nuclear volume of MCF10A cells (n=25, red), MDA-231 cells (n=29, black), and MDA-231 cells expressing GFP-emerin (n=32, blue), GFP-M45A (n=22, green), GFP-S54F (n=28, purple), GFP-M151 (n=26, yellow), and GFP-M196 (n=19, cyan). Error bars represent standard error. **p-value = 0.0030, *p-value = 0.041, one-way ANOVA. (G) Representative DAPI (blue), GFP (green), and merged images for MCF10A cells, MDA-231 cells and MDA-231 cells expressing GFP-emerin or each GFP-emerin mutant analyzed in F. Scale bars: 10 μm. (H) Nuclear circularity of MDA-231 cells (n=55, black) and MDA-231 cells expressing GFP-emerin (n=52, blue), GFP-M45A (n=53), green), GFP-S54F (n=50, purple), GFP-M151 (n=49, yellow), GFP-M24 (n=44, pink), or GFP-M196 (n=59, cyan). Error bars represent standard error. *p-value = 0.0035, **p-value = 0.0005, ***p-value = 0.0225, ****p-value < 0.0001, one-way ANOVA (I) Ratio of nuclear to cytoplasmic area in MDA-231 cells (n=61, black) and MDA-231 cells expressing GFP-emerin (n=63, blue), GFP-M45A (n=45, green), GFP-S54F (n=65, purple), GFP-M151 (n=55, yellow) or GFP-M196 (n=54, cyan). Error bars represent standard error. *p-value = 0.051, **p-value = 0.035, ****p-value < 0.0001, one-way ANOVA. (J) Nuclear area of MCF10A cells (n=65, black), MCF10A cells with no plasmid, but electroporated (electroporation control; n=45, red), and MCF10A cells expressing GFP (n=90, orange), GFP-emerin (n=76, blue), GFP-M45A (n=90, green), GFP-S54F (n=91, purple), GFP-M151 (n=72, yellow), GFP-M24 (n=72, pink) or GFP-M196 (n=72, cyan). Error bars represent standard error. **p-value = 0.0101, one-way ANOVA.

Article Snippet: Primary antibodies used for blotting included rabbit emerin polyclonal antibody (1:5,000 dilution; Proteintech, Rosemont, IL, cat#: 10351-I-AP), mouse gamma-tubulin monoclonal antibody (1:10,000 dilution; Sigma-Aldrich, cat#: T6557, RRID:AB_532292), mouse actin monoclonal antibody (1:2,500 dilution; Sigma-Aldrich, cat#: A5441, RRID:AB_476744), rabbit H3K9me2 polyclonal antibody (1:5,000 dilution; Active Motif, cat#: 39239, RRID:AB_2793199), rabbit H3K9me3 polyclonal antibody (1:10,000 dilution; Abcam, Cambridge, UK, cat#: ab8898, RRID:AB_306848), and rabbit H3K27me3 polyclonal antibody (1:10,000 dilution; MilliporeSigma, cat#: 07–449, RRID:AB_310624).

Techniques: Western Blot, Stable Transfection, Expressing, Mutagenesis, Standard Deviation, Binding Assay, Disruption, Plasmid Preparation, Electroporation, Control

Journal: Molecular cancer research : MCR

Article Title: Defects in Emerin-nucleoskeleton binding disrupt nuclear structure and promote breast cancer cell motility and metastasis

doi: 10.1158/1541-7786.MCR-20-0413

Figure Lengend Snippet: (A) Western blot quantification of H3K9me2, H3K9me3, and H3K27me3 expression in MDA-231 cells, and MDA-231 cells expressing GFP-emerin, GFP-M45A, GFP-S54F, GFP-M151, and GFP-M196. Expression was normalized to gamma-tubulin and MDA-231 cells. Error bars represent standard error. All comparisons had p-value > 0.05. (B) Representative Western blot of MDA-231 cells and MDA-231 cells expressing GFP-emerin, GFP-M45A, GFP-S54F, GFP-M151, and GFP-M196. (C) The number of cells migrating through 3.0 μm transwell pores is shown for MCF10A cells, MDA-231 cells, and MDA-231 cells expressing GFP, GFP-emerin, GFP-M45A, GFP-S54F, GFP-M151, GFP-M24 or GFP-M196. Error bars represent standard deviation. ****p-value < 0.0001, one-way ANOVA (D) Representative DAPI (blue) images for each of the cell lines analyzed in C. Scale bars: 400 μm. (E) The number of cells invading through 8.0 μm transwell pores with a Matrigel® coating was measured for MCF10A cells, MDA-231 cells, and MDA-231 cells expressing GFP-emerin, GFP-M45A, GFP-S54F, GFP-M151, or GFP-M196. Error bars represent standard deviation. *p-value = 0.0009, **p-value = 0.0005, ****p-value < 0.0001, one-way ANOVA (F) Representative DAPI (blue) images used for E. Scale Bars: 400 μm. (G) Scratch-wound healing assay. MCF10A cells, MDA-231 cells, and MDA-231 cells expressing GFP-emerin, GFP-M45A, GFP-S54F, GFP-M151, or GFP-M196 were plated, scratched with a pipette tip, and migration of cells into the wound area was monitored for 2, 4, 12 and 24 hours. Percent healed (%) refers to the ability of cells to migrate into the wound area. Error bars represent standard deviation. (H) Representative phase images for each cell line analyzed in G. Scale bars: 400 μm.

Article Snippet: Primary antibodies used for blotting included rabbit emerin polyclonal antibody (1:5,000 dilution; Proteintech, Rosemont, IL, cat#: 10351-I-AP), mouse gamma-tubulin monoclonal antibody (1:10,000 dilution; Sigma-Aldrich, cat#: T6557, RRID:AB_532292), mouse actin monoclonal antibody (1:2,500 dilution; Sigma-Aldrich, cat#: A5441, RRID:AB_476744), rabbit H3K9me2 polyclonal antibody (1:5,000 dilution; Active Motif, cat#: 39239, RRID:AB_2793199), rabbit H3K9me3 polyclonal antibody (1:10,000 dilution; Abcam, Cambridge, UK, cat#: ab8898, RRID:AB_306848), and rabbit H3K27me3 polyclonal antibody (1:10,000 dilution; MilliporeSigma, cat#: 07–449, RRID:AB_310624).

Techniques: Western Blot, Expressing, Standard Deviation, Wound Healing Assay, Transferring, Migration

Journal: Molecular cancer research : MCR

Article Title: Defects in Emerin-nucleoskeleton binding disrupt nuclear structure and promote breast cancer cell motility and metastasis

doi: 10.1158/1541-7786.MCR-20-0413

Figure Lengend Snippet: (A) Normal, non-cancerous nuclei are uniformly shaped with an organized nuclear lamina. The forces exerted on the nucleus from the cytoskeleton is countered by the forces within the nucleus, resulting in no deformations. The chromatin is compacted properly with heterochromatin tethered to the periphery and euchromatin centrally localized. (B) In a metastatic cancer cell, there is disruption of nuclear lamina proteins and significantly less emerin. This results in a disorganized nuclear lamina structure. The forces exerted on the nucleus from the cytoskeleton cannot be countered by the nucleoskeleton, resulting in a smaller, deformed nucleus that can easily migrate and metastasize. (C) When emerin is added to an invasive breast cancer cell, the nucleus can now properly organize the nucleoskeleton, which causes the nucleus to increase in size and shape. These changes block intravasation from occurring. (D) When emerin mutants that fail to bind the nucleoskeleton are added to invasive breast cancer cells, the nuclei are unable to reorganize the nuclear lamina properly. This fails to alter the nuclear morphology or size, and thus these cells can still intravasate and metastasize.

Article Snippet: Primary antibodies used for blotting included rabbit emerin polyclonal antibody (1:5,000 dilution; Proteintech, Rosemont, IL, cat#: 10351-I-AP), mouse gamma-tubulin monoclonal antibody (1:10,000 dilution; Sigma-Aldrich, cat#: T6557, RRID:AB_532292), mouse actin monoclonal antibody (1:2,500 dilution; Sigma-Aldrich, cat#: A5441, RRID:AB_476744), rabbit H3K9me2 polyclonal antibody (1:5,000 dilution; Active Motif, cat#: 39239, RRID:AB_2793199), rabbit H3K9me3 polyclonal antibody (1:10,000 dilution; Abcam, Cambridge, UK, cat#: ab8898, RRID:AB_306848), and rabbit H3K27me3 polyclonal antibody (1:10,000 dilution; MilliporeSigma, cat#: 07–449, RRID:AB_310624).

Techniques: Disruption, Blocking Assay