human mcf10a breast epithelial cells  (ATCC)

Journal: Frontiers in Pharmacology

Article Title: Extracellular vesicle-mediated preconditioning of osteoblasts by ER+ breast cancer cells: implications for the bone metastatic niche

doi: 10.3389/fphar.2026.1844422

Figure Lengend Snippet: T47D cells exhibit enhanced extracellular vesicle production. (A) Schematic illustrating extracellular vesicle (EV) biogenesis pathways, including exosome release through the endosomal-multivesicular body pathway and microvesicle (MV) shedding via outward budding from the plasma membrane. Schematic adapted from Nashed, M. (2026) on BioRender. (B) Representative histogram from nanoparticle tracking analysis (NTA) showing EV production rate and size distribution of EVs for T47D breast cancer cells and MCF10A cells as a control (n = 3 per group). Particle concentration is normalized to the max peak for each experimental group. Shaded area shows the standard error of the mean (SEM) for all histograms. (C) Maximum production rate in the exosome regime (30–150 nm) for T47D and MCF10A cells. (D) Maximum production rate in the MV regime (shown here as 150–500 nm) for T47D and MCF10A cells. All data and error bars in the bar graphs represent mean ± SEM from n = 3 independent experiments. Statistical significance was determined using an unpaired two-tailed Student’s t-test to calculate represented p -values; n.s indicates not significant.

Article Snippet: Human MCF10A breast epithelial cells (ATCC, CRL-10317) were cultured in DMEM/F12 medium (ATCC, 30–2006) supplemented with 5% horse serum (ATCC, 30–2030), human recombinant epidermal growth factor (EGF; Gibco, PHG0311) at a final concentration of 20 ng/mL, hydrocortisone (Sigma-Aldrich, H0888) at 0.5 μg/mL, cholera toxin (Sigma-Aldrich, C8052) at 100 ng/mL, and human recombinant insulin (Gibco, 12-585-014) at 10 μg/mL.

Techniques: Clinical Proteomics, Membrane, Analysis, Control, Concentration Assay, Two Tailed Test

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Knockout of Mucin 1 inhibits the proliferation, migration, and invasion of human MDA-MB-231 cells by blocking autophagy flow

doi: 10.1590/1414-431X2026e15075

Figure Lengend Snippet: Mucin 1 (MUC1) expression in human breast cancer. A , MUC1 expression levels in breast cancer (UALCAN website). B , MUC1 expression levels in different subtypes of breast cancer (UALCAN website). C , MUC1 protein expression in breast cancer (Human Protein Atlas database). D , MUC1 expression in human normal mammary epithelial cells MCF10A, TNBC cells (MDA-MB-231), and TNBC tissues (scale bar=100 μm). Data are reported as means±SE. **P<0.01, ***P<0.001, ****P<0.0001; two-tailed unpaired Student's t -test. BRCA: breast cancer gene; TCGA: The Cancer Genome Atlas; TNBC: triple-negative breast cancer.

Article Snippet: The human triple-negative breast cancer cell line MDA-MB-231 and the normal human breast epithelial cells MCF10A were obtained from the Procell Biotechnology Company (China).

Techniques: Expressing, Two Tailed Test

Journal: Oncogene

Article Title: Single amino-acid differences define H2B variants and modify chromatin accessibility to induce EMT in breast cancer

doi: 10.1038/s41388-025-03636-1

Figure Lengend Snippet: A H2B gene expression in tumors vs. adjacent normal tissues. Data are from the TCGA database, with p adj < 0.05. Gray boxes indicate no significant difference in gene expression. LFC = Log 2 (FoldChange). BRCA breast invasive carcinoma, BLCA bladder urothelial carcinoma, UCEC uterine corpus endometrial carcinoma, LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; LIHC = liver hepatocellular carcinoma; PRAD = prostate adenocarcinoma; KIRC kidney renal clear cell carcinoma, COAD colon adenocarcinoma, STAD stomach adenocarcinoma, KIRP kidney renal papillary cell carcinoma, THCA thyroid carcinoma, KICH kidney chromophobe. B Differential expression of H2B genes across breast cancer subtypes. C Kaplan–Meier plots of overall survival for patients over-expressing HIST1H2BO . p value as determined by log rank test for all analyses and Hazard ratio (HR) are provided in the graph. D Correlation between H2B variant gene expression, age, and race. E HIST1H2BC , HIST1H2BH , and HIST1H2BO expression in commonly used immortalized breast cancer cell lines relative to the non-cancerous MCF10A cell line. One-way ANOVA with Dunnett’s post-hoc test was used to assess statistical significance. Cell lines derived from patients with metastatic disease are underlined. One-way ANOVA was followed by Dunnett’s post-hoc test to assess the statistical significance in the gene expression differences observed between the breast cancer cell lines relative to the control.

Article Snippet: MCF10A human breast epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Gene Expression, Quantitative Proteomics, Expressing, Variant Assay, Derivative Assay, Control

Journal: Oncogene

Article Title: Single amino-acid differences define H2B variants and modify chromatin accessibility to induce EMT in breast cancer

doi: 10.1038/s41388-025-03636-1

Figure Lengend Snippet: A The experimental design of our in vitro cellular experiments. B Minigene system for stably expressing H2B variants in MCF10A cells. C Immunoblots show that H2B variants co-localize with H3 in the chromatin-bound sub-cellular fraction. EV: empty vector. Please note that the markers used for subcellular fractionation are shown as an example from one cell line; the full immunoblots are provided in Fig. S . D The top 30 differentially expressed genes (DEGs) in MCF10A cells that stably express HIST1H2BO . E The top enriched MSigDB C6 oncogenic signatures in MCF10A cells stably expressing HIST1H2BO .

Article Snippet: MCF10A human breast epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: In Vitro, Stable Transfection, Expressing, Western Blot, Plasmid Preparation, Fractionation

Journal: Oncogene

Article Title: Single amino-acid differences define H2B variants and modify chromatin accessibility to induce EMT in breast cancer

doi: 10.1038/s41388-025-03636-1

Figure Lengend Snippet: A CUT&RUN over-representation analysis of C6 oncogenic signatures and activating H3K4me3 marks in MCF10A cells transfected with H2B variant minigenes. Cells transfected with an empty vector (EV) served as controls. B Same as A , except for Hallmark signatures. C Microscope images of MCF10A cells stably transfected with H2B variants minigenes. An elongated, spindle-like morphology is indicative of cells undergoing the epithelial-to-mesenchymal transition (EMT). Scale bar is 200 µm. D Migrating cells 24 h after seeding in a transwell insert (n = 3; biological replicates); ****p < 0.0001. E Immunoblot of HA-tagged H2B variants and EMT markers in MCF10A cells stably transfected with H2B variant minigenes. Cells expressing HIST1H2BO expressed the highest level of mesenchymal markers.

Article Snippet: MCF10A human breast epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Transfection, Variant Assay, Plasmid Preparation, Microscopy, Stable Transfection, Western Blot, Expressing

Journal: Oncogene

Article Title: Single amino-acid differences define H2B variants and modify chromatin accessibility to induce EMT in breast cancer

doi: 10.1038/s41388-025-03636-1

Figure Lengend Snippet: A Cytokine-induced transformation of MCF10A cells, and representative microscope images of non-treated and transformed cells. Scale bar is 200 µm. B Relative expression of epithelial and mesenchymal markers in untreated and cytokine-transformed MCF10A cells (n = 3); **** p < 0.0001. C Immunoblots of epithelial and mesenchymal markers. D Relative gene expression of H2B variants in untreated and cytokine-transformed MCF10A cells (n = 3); ****p < 0.0001. E PCA plot of total RNA-seq profiles. F Top enriched C6 oncogenic signatures in MCF10A cells stably expressing H2B variant minigenes, and in cytokine-transformed cells. G , H MCF10A cells expressing HIST1H2BO have similar oncogenic gene expression patterns as cytokine-transformed cells. Each column is a MSigDB C6 oncogenic signatures; each row is a gene within the respective signature. LFC = log fold-change.

Article Snippet: MCF10A human breast epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Transformation Assay, Microscopy, Expressing, Western Blot, Gene Expression, RNA Sequencing, Stable Transfection, Variant Assay

Journal: Oncogene

Article Title: Single amino-acid differences define H2B variants and modify chromatin accessibility to induce EMT in breast cancer

doi: 10.1038/s41388-025-03636-1

Figure Lengend Snippet: A Immunoblot of H2B variant expression in untreated and cytokine-transformed cells. B Enriched Hallmark signatures in cytokine-transformed cell expressing HIST1H2BO . C Flow cytometry scatter plots and percent of transfected and cytokine-treated cells showing evidence of aneuploidy. The arrow indicates aneuploidy in the H2B1O flow cytometry data. Data in the bar plot are normalized to cells transfected with an empty vector and treated with TNFα and TGFβ. D Number of migrating cells in a transwell assay (n = 3; biological replicates). Cytokine-treatment of transfected cells increases migration relative to cytokine-treated EV control cells (see Fig. ). Response of transfected MCF10A cells to ( E ) Olaparib, ( F ) Doxoruicin, ( G ) Paciltaxel, and ( H ) Rineterkib, as measured with a colony formation assay.

Article Snippet: MCF10A human breast epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Variant Assay, Expressing, Transformation Assay, Flow Cytometry, Transfection, Plasmid Preparation, Transwell Assay, Migration, Control, Colony Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: Induction and characterization of EMT and MET in breast cancer cell lines. (A) Immunoblot analysis of EMT induction in MCF7 cells transiently overexpressing TWIST1-GFP for 48 h. Cells were transfected with 2 μg of pEGFPN1 or hTWIST1-GFP (B) Immunofluorescence analysis of EMT in MCF7 cells overexpressing TWIST1-GFP or control pEGFPN1. E-cadherin and Vimentin (red), nucleus (DAPI, blue). Scale bar ∼10 μm (C) Schematic representation of EMT induction in MCF7 cells (D) Scatter plot of immunofluorescence assay, showing relative changes in the integrated density of E-cadherin and Vimentin in MCF7 cells. Quantification for panel (B) ( n = 200). Data represent mean ± standard deviation (SD) from N = 3 independent biological replicates. (E) Immunoblot analysis of EMT induction in MCF10A cells treated with 10 ng/ml TGF-β for 7 days (F) Immunofluorescence analysis of EMT in MCF10A cells treated with TGF-β. E-cadherin (green); Vimentin (red); nucleus (blue, DAPI). Scale bar ∼10 μm (G) Schematic representation of EMT induction in MCF10A cells (H) Scatter plot of immunofluorescence assay, showing relative changes in the integrated density of E-cadherin and Vimentin in MCF10A cells [data shown in panel (F)] following EMT induction ( n = 200). Data represent mean ± SD from N = 3 independent biological replicates (I) Immunoblot analysis of MET induction in MDAMB231 cells following doxycycline-induced GRHL2 overexpression for 48 h (J) Immunofluorescence analysis of MET in MDAMB231 cells overexpressing GRHL2. E-cadherin and Vimentin (red), nucleus (blue, DAPI). Scale bar ∼10 μm (K) Schematic representation of MET induction in MDAMB231 cells (L) Scatter plot of immunofluorescence assay, showing relative changes in the integrated density of E-cadherin and Vimentin in MDAMB231 cells following MET induction [data shown in panel (J)] ( n = 200). Data represent mean ± SD from three independent biological replicates. Unpaired Student’s t -test was used to compute the P -value.

Article Snippet: Immortalized human breast epithelial cell line MCF10A (CRL-10317) and cancer cell lines MCF7 (HTB-22), and MDAMB231 (HTB-26) were obtained from ATCC.

Techniques: Western Blot, Transfection, Immunofluorescence, Control, Standard Deviation, Over Expression

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: Impact of EMT induction on Lamin A/C expression. (A, D, G) Immunofluorescence analysis of Lamin A/C (red) in MCF7 (A) , MCF10A (D) , and MDAMB231 (G) cells upon EMT (A, D) or MET (G) nucleus (DAPI, blue). Scale bar ∼10 μm. (B, E, H) Mean fluorescence intensity of Lamin A/C quantified by line scan analysis across the nucleus in MCF7 (B) , MCF10A (E) , and MDAMB231 (H) cells. Data represent mean ± SD from N = 3 independent biological replicates ( n = 250). Unpaired Student’s t -test was used to calculate P -values. (C, F, I) Immunoblot analysis of total Lamin A/C protein levels in MCF7 (C) , MCF10A (F) , and MDAMB231 (I) cells upon EMT (C, F) or MET (I) induction. GAPDH (C, F) and HSP70 (I) are loading controls. (J) Immunoblot analysis of Lamin A/C, Lamin B1, and Lamin B2 levels across 11 cell lines of breast origin with increasing mesenchymal characteristics. Loading control: Histone H3 (K) RT-qPCR analysis of LMNA transcript levels in MCF7 and MCF10A upon EMT and MET in MDAMB231 cells. Data represent mean ± SD ( N = 3, n = 9). Unpaired Student’s t -test was used to compute the P -values. Means are compared between (B) −Twist1 (control) and +Twist1; (E) −TGFβ (control) and (H) +TGFβ; −GRHL2 (control) and +GRHL2, statistical significance, P -value <0.05.

Article Snippet: Immortalized human breast epithelial cell line MCF10A (CRL-10317) and cancer cell lines MCF7 (HTB-22), and MDAMB231 (HTB-26) were obtained from ATCC.

Techniques: Expressing, Immunofluorescence, Fluorescence, Western Blot, Control, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: Effect of Lamin A/C perturbation on EMT and MET. (A) Volcano plot showing differentially expressed genes in MCF10A cells upon Lamin A/C knockdown. Downregulated, upregulated, and nonsignificant genes. (B) Heatmap of the top 50 [ P <0.05; log 2 Fold Change (FC) > 2] differentially expressed genes in MCF10A cells upon Lamin A/C knockdown. Downregulated and upregulated genes, respectively ( N = 2 biological replicates). (C) GO enrichment analysis of differentially expressed genes ( P ≤0.05), showing the most enriched biological processes. (D) GSEA plot showing enrichment for EMT upon Lamin A/C knockdown (normalized enrichment score = 3.337). (E, F) Immunofluorescence analysis of MCF7 (E) and MCF10A (F) cells upon Lamin A/C knockdown. Lamin A/C, E-cadherin or Vimentin, and phalloidin. Nucleus (DAPI). Scale bars, ∼10 μm (G) Immunofluorescence analysis of MDAMB231 cells overexpressing Lamin A*-GFP upon endogenous Lamin A/C depletion. E-cadherin (top panel) or Vimentin (bottom panel), and Lamin A/C (−Dox and +Dox panels only). Nucleus (DAPI). Scale bar ∼10 μm. ( H–J ) Immunoblot analysis of EMT markers in MCF7 (H) , MCF10A (I) cells upon Lamin A/C knockdown, and MDAMB231 (J) cells upon Lamin A overexpression. RNA-Seq was performed in N = 2 independent biological replicates. Lamin A* denotes a full-length Lamin A construct resistant to doxycycline-induced depletion of endogenous Lamin A/C. Statistical significance, P -value <0.05.

Article Snippet: Immortalized human breast epithelial cell line MCF10A (CRL-10317) and cancer cell lines MCF7 (HTB-22), and MDAMB231 (HTB-26) were obtained from ATCC.

Techniques: Knockdown, Immunofluorescence, Western Blot, Over Expression, RNA Sequencing, Construct

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: Dynamic Remodeling of the Lamin A/C Interactome During EMT and MET. (A, B) Venn diagrams showing unique and common interactors of Lamin A/C identified by (Immunoprecipitation - Mass Spectroscopy) IP-MS in MCF7 versus MCF7-TWIST1 (A) and MDAMB231 versus MDAMB231-GRHL2 (B). (C, D) Representative STRING network analysis of Lamin A/C interactors in MCF7 versus MCF7-TWIST1 (C) and MDAMB231 versus MDAMB231-GRHL2 (D) . ( E–G ) Co-IP of Lamin A/C in MCF7 (E) , MCF10A (F) , and MDAMB231 (G) cells upon EMT (E, F) or MET (G) induction, followed by immunoblotting for EZH2 and Lamin A/C. IgG: isotype control, an approximately equal amount of antibody is used for immunoprecipitation. (H, I) Proximity ligation assay (PLA) detects Lamin A/C–EZH2 interaction in MCF7 (H) and MDAMB231 (I) cells upon EMT (H) or MET (I) induction. PLA signal (red), nucleus (blue, DAPI). Scale bar: ∼10 μm. (J, K) Quantification of PLA signal in MCF7 (J) and MDAMB231 (K) cells. Data represent mean ± SD from N = 3, independent biological replicates, and P -values calculated by one-way ANOVA and means are compared between pBp-EV and pBP-Twist (J) and −Dox (GRHL2) and +Dox (GRHL2) (K). (L) Time-course analysis of Lamin A/C–EZH2 interaction by immunoprecipitation of Lamin A/C in MCF10A cells during EMT progression [∼12 to ∼168 h (∼7 days) post-TGF-β treatment] and MET recovery [5 days post-TGF-β withdrawal (WD)], assessed by Co-IP and immunoblotting. IgG: isotype control, statistical significance, P -value <0.05.

Article Snippet: Immortalized human breast epithelial cell line MCF10A (CRL-10317) and cancer cell lines MCF7 (HTB-22), and MDAMB231 (HTB-26) were obtained from ATCC.

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Co-Immunoprecipitation Assay, Western Blot, Control, Proximity Ligation Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: CDK1-mediated phosphorylation regulates Lamin A/C–EZH2 interaction and EMT progression. (A) Co-IP of FLAG in HEK293T cells co-transfected with full-length EZH2-FLAG and Lamin A-GFP deletion mutants (ΔHead 1–29, ΔRod 31–387, ΔIgG 428–549, ΔTail 550–664 of Lamin A). (B) Co-IP of GFP in HEK293T cells co-transfected with full-length Lamin A-GFP and EZH2-FLAG deletion mutants (Δ1–300, Δ301–500, Δ501–746 of EZH2). (C) PLA detects Lamin A/C–pCDK1(T161) interaction in MCF10A cells treated with 10 ng/ml TGF-β for ∼7 days. Nucleus (DAPI), PLA signal in red. Scale bar ∼10 μm (D) PLA detects Lamin A/C– EZH2 interaction in MCF10A cells treated with DMSO or 10 μM RO3306 for ∼18 h in the ± TGF-β for ∼7 days, nucleus (DAPI). PLA signal in red. Scale bar ∼10 μm. (E) Schematic representation of RO3306 and MG132 treatment in MCF7 cells (F) Quantification of PLA foci/nucleus of the data in (C) . P -values were calculated using ANOVA. Means are compared between +TGFβ and –TGFβ (control) conditions with the single antibody control. (G) Quantification of PLA foci/nucleus of the data in (D) . Statistical significance was determined using unpaired Student’s t -tests. Means are compared between +TGFβ and –TGFβ (control) conditions within each group (DMSO and RO-3306). (H) Immunofluorescence of MCF7 cells transiently transfected with pEGFP-N1 or Twist1-GFP and treated with 10 μM RO3306 for 18 h. nucleus (DAPI). Scale bar ∼10 μm (I) Immunofluorescence and quantification of colocalized voxels and Mander’s coefficient for Lamin A/C and EZH2 in MCF10A cells ± TGF-β and 1 μM MG132. Nucleus (DAPI). Scale bar ∼10 μm. Statistical significance was determined using unpaired Student’s t -tests. Means are compared between +TGFβ and –TGFβ (control) conditions within each treatment group (DMSO and RO-3306). (J) Immunofluorescence of MCF7 cells treated with MG132 and transient overexpression of hTWIST1-GFP and stained for E-cadherin and Vimentin ; nucleus (DAPI). Scale bar ∼10 μm. (K) Immunoblotting for EMT markers in MCF7 treated with RO3306 and Twist1-GFP in MCF7 (L) Immunoblotting for EMT markers in MCF7 cells treated with MG132 and Twist1-GFP in MCF7. For all experiments, data are represented as mean ± SD from N = 3 three independent biological replicates, statistical significance, P -value <0.05.

Article Snippet: Immortalized human breast epithelial cell line MCF10A (CRL-10317) and cancer cell lines MCF7 (HTB-22), and MDAMB231 (HTB-26) were obtained from ATCC.

Techniques: Phospho-proteomics, Co-Immunoprecipitation Assay, Transfection, Control, Immunofluorescence, Over Expression, Staining, Western Blot

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: Phosphorylation-dependent regulation of Lamin A/C–EZH2 binding in EMT and MET. (A, B) Schematic representation of the workflow for generating stable cell lines with inducible knockdown of Lamin A (A) or EZH2 (B) , followed by rescue with full-length, phosphodeficient, or phosphomimetic mutants. (C, D) Co-IP of Lamin A in MCF7 and MDAMB231 cells after doxycycline-induced Lamin A/C depletion and rescue with full-length, phosphodeficient (S22A), or phosphomimetic (S22D) Lamin A. TWIST1-GFP was transiently overexpressed in MCF7 cells, and GRHL2-GFP was stably overexpressed in MDAMB231 cells. (E, F) Coimmunoprecipitation of EZH2 in MCF7 and MDAMB231 cells after doxycycline-induced EZH2 depletion and rescue with full-length, phosphodeficient (T345A), or phosphomimetic (T345D) EZH2. TWIST1-GFP was transiently overexpressed in MCF7 cells, and GRHL2-GFP was stably overexpressed in MDAMB231 cells. (G) Immunofluorescence images of MCF10A cells showing the extent of colocalization between Lamin A [full-length, phosphodeficient (S22A), or phosphomimetic (S22D)] and EZH2 ± TGF-β. Nucleus (DAPI). Scale bar ∼10 μm. (H) Immunofluorescence images of MCF10A cells showing the extent of colocalization between EZH2 [full-length, phosphodeficient (T345A), or phosphomimetic (T345D)] nucleus (DAPI). Scale bar ∼10 μm. (I, J) Quantification of Lamin A and EZH2 colocalization in MCF10A cells using Mander’s coefficient. Unpaired Student’s t -test was used to compute the P -value. Means are compared between (I) LMNA-GFP (UT; control) versus LMNA-S22D (UT) and LMNA-GFP (TGFβ; control) versus LMNA-S22D (TGFβ). (J) EZH2-FLAG (UT; control) versus EZH2-T345D (UT) and EZH2-FLAG (TGFβ; control) versus EZH2-T345D (TGFβ). Statistical significance, P -value <0.05.

Article Snippet: Immortalized human breast epithelial cell line MCF10A (CRL-10317) and cancer cell lines MCF7 (HTB-22), and MDAMB231 (HTB-26) were obtained from ATCC.

Techniques: Phospho-proteomics, Binding Assay, Stable Transfection, Knockdown, Co-Immunoprecipitation Assay, Immunofluorescence, Control

Journal: Nucleic Acids Research

Article Title: Phosphorylation-dependent modulation of the Lamin A/C–EZH2 complex regulates epithelial–mesenchymal plasticity

doi: 10.1093/nar/gkaf1464

Figure Lengend Snippet: Phosphorylation-dependent regulation of EZH2 and Lamin A/C during EMT and MET. Representative mid-optical sections of immunofluorescence images showing the effect of Lamin A and EZH2 mutants on EMT (in MCF10A) and MET (in MDAMB231). MCF10A (A, C) and MDAMB231 (B, D) . Cells were transduced with full-length, phospho-deficient, or phospho-mimetic constructs of Lamin A (A, B) or EZH2 (C, D) following doxycycline-induced knockdown (0.5 μg/ml, 48 h) of endogenous Lamin A/C or EZH2. EMT was induced in MCF10A cells by TGF-β treatment (10 ng/ml, ∼7 days), while MET was induced in MDAMB231 cells by stable, constitutive overexpression of GRHL2. EZH2 was immunostained in green; E-cadherin and Vimentin were immunostained in red. Lamin A constructs were GFP-tagged. Nucleus (blue, DAPI). Scale bar ∼10 μm. (E, F) Immunoblot analysis of EMT marker expression in MCF7 and MCF10A cells upon Lamin A/C knockdown and rescue with full-length, phospho-deficient (S22A), or phospho-mimetic (S22D) Lamin A-GFP. EMT was induced by TWIST1 overexpression (∼48 h) in MCF7 cells or by 10ng/ml TGF-β (∼7 days) in MCF10A cells. (G) EM marker expression in cells with Lamin A/C knockdown was rescued with full-length, phospho-deficient (S22A) or phospho-mimetic (S22D) Lamin A/C. MET was induced by GRHL2 overexpression. (H, I) Immunoblot analysis of EMT marker expression in MCF7 and MCF10A cells upon EZH2 knockdown and rescue with full-length, phospho-deficient (T345A), or phospho-mimetic (T345D) EZH2-FLAG. EMT was induced by TGF-β (∼7 days) in MCF10A cells or by TWIST1 overexpression (∼48 h) in MCF7 cells. (J) EM marker expression in cells with EZH2 knockdown rescued with full-length, phospho-deficient (T345A), or phospho-mimetic (T345D) EZH2. MET was induced by GRHL2 overexpression.

Article Snippet: Immortalized human breast epithelial cell line MCF10A (CRL-10317) and cancer cell lines MCF7 (HTB-22), and MDAMB231 (HTB-26) were obtained from ATCC.

Techniques: Phospho-proteomics, Immunofluorescence, Transduction, Construct, Knockdown, Over Expression, Western Blot, Marker, Expressing