e cadherin  (fluidigm)

Journal: Nature

Article Title: Anti-progestin therapy targets hallmarks of breast cancer risk

doi: 10.1038/s41586-025-09684-7

Figure Lengend Snippet: a , Lobular epithelium and peri-lobular stroma (within 25 μm of the observable edge of the epithelium) were laser capture microdissected from haematoxylin and eosin-stained paired tissue sections before (baseline) and after (post-treatment) UA treatment. A representative example of undissected tissue (left) and tissue after laser ablation (right) is shown. n = 4 tissue pairs. Scale bar, 100 μm. b , Venn diagram representing the distribution of the total proteins detected (1,519) in the four participants (P06, P12, P17 and P31) used for LCM proteomics. c , Volcano plot shows differential protein abundance analysis following UA treatment. Matrisome (structural ECM or ECM-modifying) proteins among the significantly altered proteins are colour coded according to their respective subcategories. d , Gene set enrichment analysis of LCM proteomics data using the Reactome Pathways reference set, showing pathways significantly altered by UA treatment. e , Heatmap of the 27 matrisome proteins identified as significantly differentially abundant after UA treatment. ECM proteins are grouped by their structural and functional properties. f , Imaging mass cytometry was performed on paired tissue sections before (baseline) and after (post-treatment) UA treatment. Representative images show staining with metal-conjugated antibodies to E-cadherin, SOX9 and collagen VI. Nuclei were visualized using a metal-tagged DNA intercalator. The yellow boxes indicate regions corresponding to the zoomed-in inserts. n = 8 tissue pairs. Scale bars, 100 µm and 10 µm (insets). g , Single-cell neighbourhood analysis of pericellular collagen VI abundance in SOX9 high and SOX9 low cell populations across paired BC-APPS1 samples at baseline (B) and post-treatment (PT) timepoints. Tissue images were segmented into single-cell objects, and cells were classified based on expression of specific markers. Analysis was performed on E-cadherin + cells classified as either SOX9 high or SOX9 low . For each selected cell, collagen VI staining intensity was quantified within a 10-µm radius. Scale bar, 100 µm. Boxplot centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minimum and maximum values. Statistical analysis was performed using a repeated measure one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test. n = 8 tissue pairs.

Article Snippet: The antibodies used included α-smooth muscle actin (clone 1A4, 141Pr), E-cadherin (clone 24E10, 158Gd), Ki67 (clone B56, 168Er) and collagen I (polyclonal, 169Tm) from Standard BioTools (201508, Maxpar Human Immuno-oncology IMC panel kit), as well as fibronectin (polyclonal, 149Sm; ab23750, Abcam), collagen VI (polyclonal, 160Gd; ab6588, Abcam) and SOX9 (clone EPR14335 , 147Sm; 3147022D, Standard BioTools).

Techniques: Staining, Quantitative Proteomics, Functional Assay, Imaging, Mass Cytometry, Expressing

Journal: Nature

Article Title: Anti-progestin therapy targets hallmarks of breast cancer risk

doi: 10.1038/s41586-025-09684-7

Figure Lengend Snippet: A) Heatmap of the 65 proteins identified as significantly differentially abundant after UA treatment. B) Representative imaging mass cytometry images showing staining with metal-conjugated antibodies against E-Cadherin (E-Cad; luminal cell marker), α-smooth muscle actin (SMA; basal cell marker) and Collagen I (Col-I; stromal marker). E-Cad and SMA markers were used to delineate the epithelial regions and define the peri-epithelial stroma (+25 µm from epithelia). Box plots compare Col-I, Col-VI and FN1 mean intensity in epithelia and peri-epithelial stroma of paired tissue sections before (B) and after (PT) UA treatment. Box plots centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using two-sided Wilcoxon matched pairs signed rank test. N = 8 tissue pairs. C) Single-cell neighbourhood analysis of pericellular collagen I (left hand panel) and fibronectin (right hand panel) abundance for SOX9 high and SOX9 low populations across paired BC-APPS1 samples at baseline (B) and post treatment (PT) timepoints. Single-cell neighbourhood analysis was performed as described in Fig. . For each selected cell, Collagen-I or fibronectin staining intensity was quantified within a 10 µm radius. Box plots centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using a repeated measure one-way ANOVA followed by Sidak’s multiple comparisons test. N = 8 tissue pairs. D) The percentage of Ki67+ cells in epithelial SOX9 high and SOX9 low cells populations were calculated across the samples described in Extended Data Fig. 11c. Box plots centre lines represent median values, box bounds indicate the 25 th and 75 th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using repeated measure one-way ANOVA followed by Sidak’s multiple comparisons test. N = 8 tissue pairs.

Article Snippet: The antibodies used included α-smooth muscle actin (clone 1A4, 141Pr), E-cadherin (clone 24E10, 158Gd), Ki67 (clone B56, 168Er) and collagen I (polyclonal, 169Tm) from Standard BioTools (201508, Maxpar Human Immuno-oncology IMC panel kit), as well as fibronectin (polyclonal, 149Sm; ab23750, Abcam), collagen VI (polyclonal, 160Gd; ab6588, Abcam) and SOX9 (clone EPR14335 , 147Sm; 3147022D, Standard BioTools).

Techniques: Imaging, Mass Cytometry, Staining, Marker

Journal: bioRxiv

Article Title: HER2 Cellular Localization, Cell-Cell Contacts, and Cell Density Regulate Cancer Cell Plasticity in HER2+ Breast Cancer

doi: 10.1101/2023.09.28.559965

Figure Lengend Snippet: A-B ) HER2 cellular localization in low density (LD) and high density (HD) conditions in MCF10-Teti-HER2_GFP cells. Low to moderate expression of HER2 (Dox 100nM to 1 µM) resulted in similar LD images with non-membrane HER2_GFP, whereas high expression of HER2 (Dox 10 µM) led to membrane bound HER2 (A). In HD conditions, HER2 was consistently located on the cell membrane regardless of HER2 expression levels (B). C ) Immunoblotting for the nuclear fraction of MCF10A-Teti-HER2_GFP cells in LD and HD. Lamin B1 served as the loading control for the nuclear fraction, and α-Tubulin as the loading control for the cytoplasmic fraction. The absence of Tubulin in the nuclear fraction indicates the absence of cytoplasmic contamination in the nuclear fraction. D ) Protein expression levels of p-ERK, p-PDPK1, and p-AKT increase upon HER2 activation, confirming that HER2 expression can enhance cell proliferation. E-F ) Inducing HER2 expression levels increases cell proliferation, leading to higher cell density (E) and the formation of more cell clusters (F). G ) Increasing HER2 expression levels reduces the migration ability of MCF10A-Teti-HER2_GFP cells, as indicated by cell motility per frame. Cells were cultured in low density (LD) conditions (2000 cells/cm2) for 24 hours before image acquisition, and cell monitoring and image acquisition were conducted every 10 minutes over 24 hours. H ) Induction of HER2 expression levels enhances cell proliferation, subsequently leading to higher cell density and a reduction in non-membrane HER2. Cells were cultured for 72 hours in LD conditions (2000 cells/cm2) and treated with varying concentrations of Dox. The X-axis represents the final cell counts at the time of image acquisition. I ) qPCR results depict the expression levels of epithelial-mesenchymal transition (EMT) markers in MCF10A-Teti-HER2_GFP cells cultured for 72 hours in LD conditions (2000 cells/cm2) and treated with different Dox concentrations (0.2-1 µM) compared to untreated cells. J ) Immunoblotting results of MCF10A-Teti-HER2_GFP cells cultured for 72 hours in LD conditions (2000 cells/cm2) and treated with various Dox concentrations, demonstrating that HER2 expression also modulates EMT markers at the protein level. Increasing HER2 expression induces E-cadherin expression and reduces Vimentin protein levels. K ) qPCR results of EMT markers in MCF10A-Teti-HER2_GFP cells cultured for 72 hours in LD conditions (2000 cells/cm2) and treated with TGF-B (5 ng/ml daily) with or without Dox (1 µM). Notably, HER2 expression alone could not reverse the mesenchymal phenotype. L ) Prolonged culturing of cells in LD conditions intensifies the induction of the mesenchymal state in MCF10A-Teti-HER2_GFP cells. P-values in panels G and I were calculated using a two-way ANOVA test. P-values in panel L were calculated using student paired t-test between days 3 and 15. P-values in panels E and F were calculated using the Mann-Whitney U test. The p-value in panel H is presented from the Pearson correlation test. Data in panels E, F, and G are represented as medians.

Article Snippet: The antibody cocktail consisted of anti-PanKeratin antibody (clone AE-1/AE-3, 1:800, Standard BioTools, San Francisco, CA, USA), anti-vimentin (clone D21H3, 1:400, Standard BioTools), anti-gamma-catenin (clone 15F11, 1:100, BioLegend, San Diego, California, USA), anti-ALDH1 (clone 44/ALDH, 1:100, Bd Bioscience, San Jose, California, USA), anti-E cadherin (clone 24E10, 1:200, Standard BioTools), anti-HER2 (clone D8F12, 1:150, Cell Signaling Technology, Danvers, MA, USA), anti-Ki67 (clone B56, 1:100, Standard BioTools), and anti-Zeb1 (clone E2G6Y, 1:50, Cell Signaling Technology) dissolved in 0.5% BSA PBS solution.

Techniques: Expressing, Membrane, Western Blot, Control, Activation Assay, Migration, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: HER2 Cellular Localization, Cell-Cell Contacts, and Cell Density Regulate Cancer Cell Plasticity in HER2+ Breast Cancer

doi: 10.1101/2023.09.28.559965

Figure Lengend Snippet: A ) Cell-cell contact is essential for HER2 membrane localization and stabilization. HER2 is expressed on the cell membrane when two cells make contact. B ) HER2 staining in a primary breast tumor (HER2 IHC scores 1+, 2+, 3+). Arrows indicate solitary cancer cells separated from the tumor mass structure with different HER2 cellular localization compared to the cells in the tumor mass. The right graph quantifies HER2 non-membrane localization and the number of cell contacts. An increase in the number of cell-cell contacts (an indicator of cell density) leads to increased membrane HER2 localization. C ) SKBR3 cells cultured in non-adherent conditions and shaken for 48 hours to inhibit cell contact. HER2 disappeared from the membrane after 48 hours of shaking. D ) Quantification of the intensity signal of HER2 staining in Shaking-SKBR3 cells after 24, 48, and 72 hours. E ) HER2 staining (Cy3) in SKBR3 cells, seeded after 48 hours of shaking in LD condition and fixed after 6 hours. Quantification shows the correlation between HER2 staining signal (Cy3) and DAPI in the nuclear and endoplasmic reticulum area (NER). F ) Mass spectrometric analysis identified desmosome junction components, particularly Desmoplakin (DSP) and Vimentin (VIM), as consistent binding partners of HER2 across LD and HD conditions in MCF10A-teti-HER2_GFP cells and shaking-SKBR3 cells. G ) The physical binding of HER2 to Vim and DSP was further confirmed by co-immunoprecipitation in MCF10A-Teti-HER2-GPF cells treated with 1 µM Dox. H-I ) Co-culture of MCF10A-teti-HER2_GFP cells with different cell types and tracking cell-cell contact and HER2 membrane localization. MCF10A-teti-HER2_GFP cells were cultured in LD condition for 24 hours and then co-cultured (in the same well as mixed culture) for an additional 24 hours with different cell types and extracellular matrix (ECM) conditions. Certain epithelial cell lines such as SKBR3 and MDA-MB453 could restore HER2 to the membrane, while others (such as HEK293T and SV80) did not exhibit this ability. The quantification represents the percentage of cell contacts overlapping with HER2 membrane localization in MCF10A-teti-HER2_GFP cells. J ) mRNA expression of desmosome adhesion surface cadherin proteins, which show higher cadherin expression values for cells that can facilitate HER2 membrane localization in the co-cultures with MCF10A-teti-HER2_GFP. K ) Upon siRNA knockdown of DSP in MCF10A-Teti-HER2-GPF cells, the total number of cells decreased in the KD group compared to the control (Scramble). This reduction in the number of cells appears to be due to a decreased cell junction ability in the DSP-KD group, as demonstrated in the right graph by increasing number of single cells in DSP-KD group. L ) The intensity of HER2 and DSG2 decreased on the cell contact surface of cells upon the knockdown of DSP. Cells were fixed and stained for DSG2, 6 hours after culturing in the LD condition. P-values in panels B, D, E, and I were calculated using the Mann-Whitney U test, and data are presented as medians. P-values in panel K were calculated by the Student t-test, and data are presented as means ± SD. P-values in panel J were calculated using a One-way ANOVA test. P-values in panel L were calculated by the chi-square test.

Article Snippet: The antibody cocktail consisted of anti-PanKeratin antibody (clone AE-1/AE-3, 1:800, Standard BioTools, San Francisco, CA, USA), anti-vimentin (clone D21H3, 1:400, Standard BioTools), anti-gamma-catenin (clone 15F11, 1:100, BioLegend, San Diego, California, USA), anti-ALDH1 (clone 44/ALDH, 1:100, Bd Bioscience, San Jose, California, USA), anti-E cadherin (clone 24E10, 1:200, Standard BioTools), anti-HER2 (clone D8F12, 1:150, Cell Signaling Technology, Danvers, MA, USA), anti-Ki67 (clone B56, 1:100, Standard BioTools), and anti-Zeb1 (clone E2G6Y, 1:50, Cell Signaling Technology) dissolved in 0.5% BSA PBS solution.

Techniques: Membrane, Staining, Cell Culture, Binding Assay, Immunoprecipitation, Co-Culture Assay, Expressing, Knockdown, Control, MANN-WHITNEY

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet:

Article Snippet: Anti-human CDH1/E-Cadherin (rabbit IgG, clone 24E10) - 158Gd , Fluidigm , Cat# 3158029D; RRID: AB_2893074.

Techniques: Conjugation Assay, Purification, Blocking Assay, Recombinant, Control, Virus, Saline, Modification, Staining, Library Quantification, Antibody Labeling, Flow Cytometry, Software, Cytometry, Sequencing

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet:

Article Snippet: Anti-human CDH1/E-Cadherin (rabbit IgG, clone 24E10) - 158Gd , Fluidigm , Cat# 3158029D; RRID: AB_2893074.

Techniques: Conjugation Assay, Purification, Blocking Assay, Recombinant, Control, Virus, Saline, Modification, Staining, Library Quantification, Antibody Labeling, Flow Cytometry, Software, Cytometry, Sequencing