human breast epithelial cell line mcf10a  (ATCC)

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

Journal: Translational Oncology

Article Title: Aurintricarboxylic acid, an inhibitor of cystathionine γ-lyase, enhances the sensitivity of chemotherapy drugs in TNBC

doi: 10.1016/j.tranon.2025.102602

Figure Lengend Snippet: The expression of CSE is negatively related to the sensitivity of chemotherapy drug in TNBC cells. (A and B) Relative expression of CSE protein in human TNBC cell lines was significantly higher compared to Hs578bst cells. ** P < 0.01. (C) mRNA level of CSE in human TNBC cell lines was significantly elevated compared to Hs578bst cells. **P<0.01. (D and E) MDA-MB-231 and BT549 cells with different levels of CSE exhibited varying sensitivities to DDP/PTX treatment. (F and G) Relative expression of P-gp protein in human TNBC cell lines was significantly higher compared to Hs578bst cells. * P < 0.05. MDA-MB-231, MDA-MB-468, BT549 were the human TNBC cell lines; Hs578bst represents normal mammary epithelial cells. All data are presented as mean ± SD ( n = 3).

Article Snippet: Human TNBC cell lines MDA-MB-231, MDA-MB-468, and human normal breast epithelial cell line Hs578Bst were purchased from the ATCC (Manassas, VA, USA).

Techniques: Expressing