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non transformed mammary cell line mcf10a  (ATCC)
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ATCC non transformed mammary cell line mcf10a
Non Transformed Mammary Cell Line Mcf10a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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epithelial cell line mcf10a  (ATCC)
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ATCC epithelial cell line mcf10a
Epithelial Cell Line Mcf10a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human breast epithelial cell line mcf10a  (ATCC)
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ATCC human breast epithelial cell line mcf10a
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 <t>MCF10A</t> 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.
Human Breast Epithelial Cell Line Mcf10a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cell lines mcf10a atcc  (ATCC)
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ATCC cell lines mcf10a atcc
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 <t>MCF10A</t> 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.
Cell Lines Mcf10a Atcc, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mammary gland derived cell lines mcf10a  (ATCC)
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ATCC mammary gland derived cell lines mcf10a
MDA-MB-231, MCF7, and <t>MCF10A</t> cells were treated for 48 h with 2.5-80 µM of brazilin, brazilin-(OMe) 3 , or brazilin-(OAc) 3 . Cell viability was assessed using an MTT assay (0.5 mg/mL). Staurosporine (St, 50 nM) served as a positive control. Viability is expressed as a percentage relative to untreated control cells (Ctrl) for (a) MDA-MB-231, (b) MCF7, and (c) MCF10A. Data represent mean ± SD from three independent biological replicates. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.
Mammary Gland Derived Cell Lines Mcf10a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human mammary epithelial cell line mcf10a  (ATCC)
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ATCC human mammary epithelial cell line mcf10a
NCT-58 exerts anti-proliferative effect in TNBC cells by targeting the C-terminal domain of HSP90. (A) Four TNBC cell lines, MDA-MB-231, BT549, Hs578T and 4T1 cells were treated with various concentrations of NCT-58 (0–20 µM) for 72 h. Cell viability was assessed using MTS assay, and IC 50 values were calculated using non-linear regression with a sigmoidal dose-response curve. (B) MDA-MB-231 and 4T1 cells were treated at the indicated concentrations of NCT-58 (0–10 µM) for 72 h. Apoptosis was determined through sub-G1-DNA analysis using flow cytometry. (C) Immunoblot analyses of PARP, cleaved-PARP, caspase-3, cleaved caspase-3, caspase-7 and cleaved caspase-7 protein expression in MDA-MB-231 cells after treatment with NCT-58 (0–10 µM, 72 h). GAPDH was used as an internal loading control. Quantitative graphs of these protein levels. The results are presented as the mean ± SEM of at least three independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post hoc test. (D) Effect of NCT-58 on C-terminal HSP90 binding activity. The inhibitory effect of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 500 µM) on the interaction between HSP90α (C-terminal) and its co-chaperone peptidylprolyl isomerase was determined using an HSP90α (C-terminal) inhibitor screening assay. (E) Influence of NCT-58 on N-terminal HSP90 binding activity. The competitive HSP90α binding activity of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 1 µM) with FITC-labeled geldanamycin was determined using an HSP90α N-terminal domain assay. (F and G) Molecular docking analysis of NCT-58 binding to the CTD of HSP90 (PDB ID: 7RY1). (F) The binding pose of NCT-58 at the dimerization interface is displayed as a space-filling model. The α1 chain of HSP90 is rendered in a blue ribbon, and the α2 chain in a pink ribbon. Connolly surface representation of the HSP90 CTD, with NCT-58 modeled within the binding interface (docking score=−9.5). (G) A 2D interaction diagram of NCT-58 with key residues in the HSP90 CTD. Hydrogen bonds and π-anion interactions are indicated by dashed green and blue lines, respectively. (H and I) Comparison of the effects of NCT-58 and the N-terminal HSP90 inhibitor geldanamycin on HSF-1 and HSP70 expression. MDA-MB-231 cells were treated with NCT-58 (300 nM and 10 µM) or geldanamycin (300 nM) for 24 h. Cells were immuno-stained for HSF-1 (red, H) or HSP70 (green, I) using DAPI (nuclei, blue). Images were acquired using a confocal microscope, and quantification of immunofluorescence intensity was performed using ImageJ software. Nuclear HSF1 intensity was expressed as the HSF1/DAPI ratio, and cytoplasmic HSP70 intensity was expressed as corrected total cell fluorescence normalized to DAPI. (J and K) Effect of NCT-58 and geldanamycin on cytotoxicity in non-malignant cells. Normal human mammary epithelial <t>MCF10A</t> (J) and 293 (K) cells were treated with various concentrations (0.1–10 µM) of NCT-58 or geldanamycin for 72 h. Cell viability was determined using MTS assay (***P<0.001). *P < 0.05, **P<0.01, ***P<0.001 and ****P<0.0001. TNBC, triple-negative breast cancer; Gelda, geldanamycin; Novo, novobiocin; CTD, C-terminal domain.
Human Mammary Epithelial Cell Line Mcf10a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mcf10a breast tissue cell lines  (ATCC)
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ATCC mcf10a breast tissue cell lines
NCT-58 exerts anti-proliferative effect in TNBC cells by targeting the C-terminal domain of HSP90. (A) Four TNBC cell lines, MDA-MB-231, BT549, Hs578T and 4T1 cells were treated with various concentrations of NCT-58 (0–20 µM) for 72 h. Cell viability was assessed using MTS assay, and IC 50 values were calculated using non-linear regression with a sigmoidal dose-response curve. (B) MDA-MB-231 and 4T1 cells were treated at the indicated concentrations of NCT-58 (0–10 µM) for 72 h. Apoptosis was determined through sub-G1-DNA analysis using flow cytometry. (C) Immunoblot analyses of PARP, cleaved-PARP, caspase-3, cleaved caspase-3, caspase-7 and cleaved caspase-7 protein expression in MDA-MB-231 cells after treatment with NCT-58 (0–10 µM, 72 h). GAPDH was used as an internal loading control. Quantitative graphs of these protein levels. The results are presented as the mean ± SEM of at least three independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post hoc test. (D) Effect of NCT-58 on C-terminal HSP90 binding activity. The inhibitory effect of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 500 µM) on the interaction between HSP90α (C-terminal) and its co-chaperone peptidylprolyl isomerase was determined using an HSP90α (C-terminal) inhibitor screening assay. (E) Influence of NCT-58 on N-terminal HSP90 binding activity. The competitive HSP90α binding activity of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 1 µM) with FITC-labeled geldanamycin was determined using an HSP90α N-terminal domain assay. (F and G) Molecular docking analysis of NCT-58 binding to the CTD of HSP90 (PDB ID: 7RY1). (F) The binding pose of NCT-58 at the dimerization interface is displayed as a space-filling model. The α1 chain of HSP90 is rendered in a blue ribbon, and the α2 chain in a pink ribbon. Connolly surface representation of the HSP90 CTD, with NCT-58 modeled within the binding interface (docking score=−9.5). (G) A 2D interaction diagram of NCT-58 with key residues in the HSP90 CTD. Hydrogen bonds and π-anion interactions are indicated by dashed green and blue lines, respectively. (H and I) Comparison of the effects of NCT-58 and the N-terminal HSP90 inhibitor geldanamycin on HSF-1 and HSP70 expression. MDA-MB-231 cells were treated with NCT-58 (300 nM and 10 µM) or geldanamycin (300 nM) for 24 h. Cells were immuno-stained for HSF-1 (red, H) or HSP70 (green, I) using DAPI (nuclei, blue). Images were acquired using a confocal microscope, and quantification of immunofluorescence intensity was performed using ImageJ software. Nuclear HSF1 intensity was expressed as the HSF1/DAPI ratio, and cytoplasmic HSP70 intensity was expressed as corrected total cell fluorescence normalized to DAPI. (J and K) Effect of NCT-58 and geldanamycin on cytotoxicity in non-malignant cells. Normal human mammary epithelial <t>MCF10A</t> (J) and 293 (K) cells were treated with various concentrations (0.1–10 µM) of NCT-58 or geldanamycin for 72 h. Cell viability was determined using MTS assay (***P<0.001). *P < 0.05, **P<0.01, ***P<0.001 and ****P<0.0001. TNBC, triple-negative breast cancer; Gelda, geldanamycin; Novo, novobiocin; CTD, C-terminal domain.
Mcf10a Breast Tissue Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mcf10a breast tissue cell lines - by Bioz Stars, 2026-03
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normal mammary epithelial cell line mcf10a  (ATCC)
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ATCC normal mammary epithelial cell line mcf10a
NCT-58 exerts anti-proliferative effect in TNBC cells by targeting the C-terminal domain of HSP90. (A) Four TNBC cell lines, MDA-MB-231, BT549, Hs578T and 4T1 cells were treated with various concentrations of NCT-58 (0–20 µM) for 72 h. Cell viability was assessed using MTS assay, and IC 50 values were calculated using non-linear regression with a sigmoidal dose-response curve. (B) MDA-MB-231 and 4T1 cells were treated at the indicated concentrations of NCT-58 (0–10 µM) for 72 h. Apoptosis was determined through sub-G1-DNA analysis using flow cytometry. (C) Immunoblot analyses of PARP, cleaved-PARP, caspase-3, cleaved caspase-3, caspase-7 and cleaved caspase-7 protein expression in MDA-MB-231 cells after treatment with NCT-58 (0–10 µM, 72 h). GAPDH was used as an internal loading control. Quantitative graphs of these protein levels. The results are presented as the mean ± SEM of at least three independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post hoc test. (D) Effect of NCT-58 on C-terminal HSP90 binding activity. The inhibitory effect of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 500 µM) on the interaction between HSP90α (C-terminal) and its co-chaperone peptidylprolyl isomerase was determined using an HSP90α (C-terminal) inhibitor screening assay. (E) Influence of NCT-58 on N-terminal HSP90 binding activity. The competitive HSP90α binding activity of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 1 µM) with FITC-labeled geldanamycin was determined using an HSP90α N-terminal domain assay. (F and G) Molecular docking analysis of NCT-58 binding to the CTD of HSP90 (PDB ID: 7RY1). (F) The binding pose of NCT-58 at the dimerization interface is displayed as a space-filling model. The α1 chain of HSP90 is rendered in a blue ribbon, and the α2 chain in a pink ribbon. Connolly surface representation of the HSP90 CTD, with NCT-58 modeled within the binding interface (docking score=−9.5). (G) A 2D interaction diagram of NCT-58 with key residues in the HSP90 CTD. Hydrogen bonds and π-anion interactions are indicated by dashed green and blue lines, respectively. (H and I) Comparison of the effects of NCT-58 and the N-terminal HSP90 inhibitor geldanamycin on HSF-1 and HSP70 expression. MDA-MB-231 cells were treated with NCT-58 (300 nM and 10 µM) or geldanamycin (300 nM) for 24 h. Cells were immuno-stained for HSF-1 (red, H) or HSP70 (green, I) using DAPI (nuclei, blue). Images were acquired using a confocal microscope, and quantification of immunofluorescence intensity was performed using ImageJ software. Nuclear HSF1 intensity was expressed as the HSF1/DAPI ratio, and cytoplasmic HSP70 intensity was expressed as corrected total cell fluorescence normalized to DAPI. (J and K) Effect of NCT-58 and geldanamycin on cytotoxicity in non-malignant cells. Normal human mammary epithelial <t>MCF10A</t> (J) and 293 (K) cells were treated with various concentrations (0.1–10 µM) of NCT-58 or geldanamycin for 72 h. Cell viability was determined using MTS assay (***P<0.001). *P < 0.05, **P<0.01, ***P<0.001 and ****P<0.0001. TNBC, triple-negative breast cancer; Gelda, geldanamycin; Novo, novobiocin; CTD, C-terminal domain.
Normal Mammary Epithelial Cell Line Mcf10a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/normal mammary epithelial cell line mcf10a/product/ATCC
Average 99 stars, based on 1 article reviews
normal mammary epithelial cell line mcf10a - by Bioz Stars, 2026-03
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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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

MDA-MB-231, MCF7, and MCF10A cells were treated for 48 h with 2.5-80 µM of brazilin, brazilin-(OMe) 3 , or brazilin-(OAc) 3 . Cell viability was assessed using an MTT assay (0.5 mg/mL). Staurosporine (St, 50 nM) served as a positive control. Viability is expressed as a percentage relative to untreated control cells (Ctrl) for (a) MDA-MB-231, (b) MCF7, and (c) MCF10A. Data represent mean ± SD from three independent biological replicates. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: bioRxiv

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and its Semi-synthetic Derivates on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.64898/2026.01.09.698710

Figure Lengend Snippet: MDA-MB-231, MCF7, and MCF10A cells were treated for 48 h with 2.5-80 µM of brazilin, brazilin-(OMe) 3 , or brazilin-(OAc) 3 . Cell viability was assessed using an MTT assay (0.5 mg/mL). Staurosporine (St, 50 nM) served as a positive control. Viability is expressed as a percentage relative to untreated control cells (Ctrl) for (a) MDA-MB-231, (b) MCF7, and (c) MCF10A. Data represent mean ± SD from three independent biological replicates. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317TM), MDA-MB-231 (CRM-HTB-26TM), and MCF7 (HTB-22TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: MTT Assay, Positive Control, Control

Proliferation of (a) MDA-MB-231, (b) MCF7, and (c) MCF10A cells treated with 20 µM of brazilin or derivatives was assessed by trypan blue cell counting at 48, 72, and 96 h. Data are expressed as fold change in cell number over time and shown as mean ± SE; mean values at 96 h are indicated. See Supp for complete cell growth data at 2.5, 5, and 40 µM. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. (d) Representative brightfield images of cells after 96 h of treatment (20 µM) captured using a NIKON® ECLIPSE Ts2 microscope. Scale bar = 200 µm. See Supp for images at 48 and 72 h. Data represent three independent biological replicates.

Journal: bioRxiv

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and its Semi-synthetic Derivates on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.64898/2026.01.09.698710

Figure Lengend Snippet: Proliferation of (a) MDA-MB-231, (b) MCF7, and (c) MCF10A cells treated with 20 µM of brazilin or derivatives was assessed by trypan blue cell counting at 48, 72, and 96 h. Data are expressed as fold change in cell number over time and shown as mean ± SE; mean values at 96 h are indicated. See Supp for complete cell growth data at 2.5, 5, and 40 µM. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. (d) Representative brightfield images of cells after 96 h of treatment (20 µM) captured using a NIKON® ECLIPSE Ts2 microscope. Scale bar = 200 µm. See Supp for images at 48 and 72 h. Data represent three independent biological replicates.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317TM), MDA-MB-231 (CRM-HTB-26TM), and MCF7 (HTB-22TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Cell Counting, Microscopy

Representative immunoblots (top) and quantification (bottom) of active cleaved caspase 3 and cleaved PARP in (a) MDA-MB-231, (b) MCF7, and (c) MCF10A cells treated with 20 µM of brazilin or derivatives for 48 h. Staurosporine (50 nM) was used as a positive control for apoptosis. GAPDH was used as a loading control. Data are presented as mean ± SE from independent biological replicates. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: bioRxiv

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and its Semi-synthetic Derivates on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.64898/2026.01.09.698710

Figure Lengend Snippet: Representative immunoblots (top) and quantification (bottom) of active cleaved caspase 3 and cleaved PARP in (a) MDA-MB-231, (b) MCF7, and (c) MCF10A cells treated with 20 µM of brazilin or derivatives for 48 h. Staurosporine (50 nM) was used as a positive control for apoptosis. GAPDH was used as a loading control. Data are presented as mean ± SE from independent biological replicates. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317TM), MDA-MB-231 (CRM-HTB-26TM), and MCF7 (HTB-22TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Positive Control, Control

Confocal images of (a) MDA-MB-231, (b) MCF7, and (c) MCF10A cells treated with 20 µM of brazilin, brazilin-(OMe) 3 , or brazilin-(OAc) 3 for 48 h. DNA damage is indicated by γH2AX (phospo-S139 H2AXc) immunostaining (red), and chromatin condensation is visualized by intense DAPI staining (blue). Scale bar = 14-19 µm (see individual images). Images were acquired using a Leica SP8 confocal microscope with Leica Application Suite X. Representative images from at least three independent experiments.

Journal: bioRxiv

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and its Semi-synthetic Derivates on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.64898/2026.01.09.698710

Figure Lengend Snippet: Confocal images of (a) MDA-MB-231, (b) MCF7, and (c) MCF10A cells treated with 20 µM of brazilin, brazilin-(OMe) 3 , or brazilin-(OAc) 3 for 48 h. DNA damage is indicated by γH2AX (phospo-S139 H2AXc) immunostaining (red), and chromatin condensation is visualized by intense DAPI staining (blue). Scale bar = 14-19 µm (see individual images). Images were acquired using a Leica SP8 confocal microscope with Leica Application Suite X. Representative images from at least three independent experiments.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317TM), MDA-MB-231 (CRM-HTB-26TM), and MCF7 (HTB-22TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Immunostaining, Staining, Microscopy

(a) Radical scavenging activity of brazilin, brazilin-(OMe) 3 , and brazilin-(OAc) 3 (2.5-80 µM) was assessed using DPPH synthetic radicals. (b) Cytoplasmic ROS levels were measured in MDA-MB-231, MCF7, and MCF10A cells after 48 h treatment with 20 µM of each compound by fluorescent detection of DHE oxidation. (c-h) Mitochondrial ROS (MitoSOX) and mitochondrial membrane potential (TMRE) were evaluated in (c,f) MDA-MB-231, (d,g) MCF7, and (e,h) MCF10A cells treated for 48 h with 20 µM or 40 µM of brazilin or derivatives. FCCP was used as a positive control for mitochondrial depolarization. Fluorescence intensities are plotted as mean ± SE relative to untreated cells for each concentration. *p<0.05, **p<0.001, ***p<0.001. Data represents three independent biological replicates.

Journal: bioRxiv

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and its Semi-synthetic Derivates on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.64898/2026.01.09.698710

Figure Lengend Snippet: (a) Radical scavenging activity of brazilin, brazilin-(OMe) 3 , and brazilin-(OAc) 3 (2.5-80 µM) was assessed using DPPH synthetic radicals. (b) Cytoplasmic ROS levels were measured in MDA-MB-231, MCF7, and MCF10A cells after 48 h treatment with 20 µM of each compound by fluorescent detection of DHE oxidation. (c-h) Mitochondrial ROS (MitoSOX) and mitochondrial membrane potential (TMRE) were evaluated in (c,f) MDA-MB-231, (d,g) MCF7, and (e,h) MCF10A cells treated for 48 h with 20 µM or 40 µM of brazilin or derivatives. FCCP was used as a positive control for mitochondrial depolarization. Fluorescence intensities are plotted as mean ± SE relative to untreated cells for each concentration. *p<0.05, **p<0.001, ***p<0.001. Data represents three independent biological replicates.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317TM), MDA-MB-231 (CRM-HTB-26TM), and MCF7 (HTB-22TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activity Assay, Membrane, Positive Control, Fluorescence, Concentration Assay

Differentially expressed genes (DEG) identified by RNA-seq in MDA-MB-231 cells. (a) ATF3 , (b) H2BC21 , (c) SOX4 , (d) MTRNR2L8 , (e) MTRNR2L1 , (f) MTRNR2L2 , and (g) GCLM , were validated by RT-qPCR in MDA-MB-231, MCF7, and MCF10A cells treated with 20 µM brazilin or derivatives for 48 h. Data are expressed as mean ± SE from three independent biological replicates. *p < 0.05, **p < 0.001, ***p < 0.001.

Journal: bioRxiv

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and its Semi-synthetic Derivates on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.64898/2026.01.09.698710

Figure Lengend Snippet: Differentially expressed genes (DEG) identified by RNA-seq in MDA-MB-231 cells. (a) ATF3 , (b) H2BC21 , (c) SOX4 , (d) MTRNR2L8 , (e) MTRNR2L1 , (f) MTRNR2L2 , and (g) GCLM , were validated by RT-qPCR in MDA-MB-231, MCF7, and MCF10A cells treated with 20 µM brazilin or derivatives for 48 h. Data are expressed as mean ± SE from three independent biological replicates. *p < 0.05, **p < 0.001, ***p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317TM), MDA-MB-231 (CRM-HTB-26TM), and MCF7 (HTB-22TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: RNA Sequencing, Quantitative RT-PCR

NCT-58 exerts anti-proliferative effect in TNBC cells by targeting the C-terminal domain of HSP90. (A) Four TNBC cell lines, MDA-MB-231, BT549, Hs578T and 4T1 cells were treated with various concentrations of NCT-58 (0–20 µM) for 72 h. Cell viability was assessed using MTS assay, and IC 50 values were calculated using non-linear regression with a sigmoidal dose-response curve. (B) MDA-MB-231 and 4T1 cells were treated at the indicated concentrations of NCT-58 (0–10 µM) for 72 h. Apoptosis was determined through sub-G1-DNA analysis using flow cytometry. (C) Immunoblot analyses of PARP, cleaved-PARP, caspase-3, cleaved caspase-3, caspase-7 and cleaved caspase-7 protein expression in MDA-MB-231 cells after treatment with NCT-58 (0–10 µM, 72 h). GAPDH was used as an internal loading control. Quantitative graphs of these protein levels. The results are presented as the mean ± SEM of at least three independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post hoc test. (D) Effect of NCT-58 on C-terminal HSP90 binding activity. The inhibitory effect of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 500 µM) on the interaction between HSP90α (C-terminal) and its co-chaperone peptidylprolyl isomerase was determined using an HSP90α (C-terminal) inhibitor screening assay. (E) Influence of NCT-58 on N-terminal HSP90 binding activity. The competitive HSP90α binding activity of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 1 µM) with FITC-labeled geldanamycin was determined using an HSP90α N-terminal domain assay. (F and G) Molecular docking analysis of NCT-58 binding to the CTD of HSP90 (PDB ID: 7RY1). (F) The binding pose of NCT-58 at the dimerization interface is displayed as a space-filling model. The α1 chain of HSP90 is rendered in a blue ribbon, and the α2 chain in a pink ribbon. Connolly surface representation of the HSP90 CTD, with NCT-58 modeled within the binding interface (docking score=−9.5). (G) A 2D interaction diagram of NCT-58 with key residues in the HSP90 CTD. Hydrogen bonds and π-anion interactions are indicated by dashed green and blue lines, respectively. (H and I) Comparison of the effects of NCT-58 and the N-terminal HSP90 inhibitor geldanamycin on HSF-1 and HSP70 expression. MDA-MB-231 cells were treated with NCT-58 (300 nM and 10 µM) or geldanamycin (300 nM) for 24 h. Cells were immuno-stained for HSF-1 (red, H) or HSP70 (green, I) using DAPI (nuclei, blue). Images were acquired using a confocal microscope, and quantification of immunofluorescence intensity was performed using ImageJ software. Nuclear HSF1 intensity was expressed as the HSF1/DAPI ratio, and cytoplasmic HSP70 intensity was expressed as corrected total cell fluorescence normalized to DAPI. (J and K) Effect of NCT-58 and geldanamycin on cytotoxicity in non-malignant cells. Normal human mammary epithelial MCF10A (J) and 293 (K) cells were treated with various concentrations (0.1–10 µM) of NCT-58 or geldanamycin for 72 h. Cell viability was determined using MTS assay (***P<0.001). *P < 0.05, **P<0.01, ***P<0.001 and ****P<0.0001. TNBC, triple-negative breast cancer; Gelda, geldanamycin; Novo, novobiocin; CTD, C-terminal domain.

Journal: Oncology Reports

Article Title: C-terminal HSP90 inhibitor NCT-58 impairs the cancer stem-like phenotype and enhances chemotherapy efficacy in TNBC

doi: 10.3892/or.2025.9018

Figure Lengend Snippet: NCT-58 exerts anti-proliferative effect in TNBC cells by targeting the C-terminal domain of HSP90. (A) Four TNBC cell lines, MDA-MB-231, BT549, Hs578T and 4T1 cells were treated with various concentrations of NCT-58 (0–20 µM) for 72 h. Cell viability was assessed using MTS assay, and IC 50 values were calculated using non-linear regression with a sigmoidal dose-response curve. (B) MDA-MB-231 and 4T1 cells were treated at the indicated concentrations of NCT-58 (0–10 µM) for 72 h. Apoptosis was determined through sub-G1-DNA analysis using flow cytometry. (C) Immunoblot analyses of PARP, cleaved-PARP, caspase-3, cleaved caspase-3, caspase-7 and cleaved caspase-7 protein expression in MDA-MB-231 cells after treatment with NCT-58 (0–10 µM, 72 h). GAPDH was used as an internal loading control. Quantitative graphs of these protein levels. The results are presented as the mean ± SEM of at least three independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post hoc test. (D) Effect of NCT-58 on C-terminal HSP90 binding activity. The inhibitory effect of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 500 µM) on the interaction between HSP90α (C-terminal) and its co-chaperone peptidylprolyl isomerase was determined using an HSP90α (C-terminal) inhibitor screening assay. (E) Influence of NCT-58 on N-terminal HSP90 binding activity. The competitive HSP90α binding activity of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 1 µM) with FITC-labeled geldanamycin was determined using an HSP90α N-terminal domain assay. (F and G) Molecular docking analysis of NCT-58 binding to the CTD of HSP90 (PDB ID: 7RY1). (F) The binding pose of NCT-58 at the dimerization interface is displayed as a space-filling model. The α1 chain of HSP90 is rendered in a blue ribbon, and the α2 chain in a pink ribbon. Connolly surface representation of the HSP90 CTD, with NCT-58 modeled within the binding interface (docking score=−9.5). (G) A 2D interaction diagram of NCT-58 with key residues in the HSP90 CTD. Hydrogen bonds and π-anion interactions are indicated by dashed green and blue lines, respectively. (H and I) Comparison of the effects of NCT-58 and the N-terminal HSP90 inhibitor geldanamycin on HSF-1 and HSP70 expression. MDA-MB-231 cells were treated with NCT-58 (300 nM and 10 µM) or geldanamycin (300 nM) for 24 h. Cells were immuno-stained for HSF-1 (red, H) or HSP70 (green, I) using DAPI (nuclei, blue). Images were acquired using a confocal microscope, and quantification of immunofluorescence intensity was performed using ImageJ software. Nuclear HSF1 intensity was expressed as the HSF1/DAPI ratio, and cytoplasmic HSP70 intensity was expressed as corrected total cell fluorescence normalized to DAPI. (J and K) Effect of NCT-58 and geldanamycin on cytotoxicity in non-malignant cells. Normal human mammary epithelial MCF10A (J) and 293 (K) cells were treated with various concentrations (0.1–10 µM) of NCT-58 or geldanamycin for 72 h. Cell viability was determined using MTS assay (***P<0.001). *P < 0.05, **P<0.01, ***P<0.001 and ****P<0.0001. TNBC, triple-negative breast cancer; Gelda, geldanamycin; Novo, novobiocin; CTD, C-terminal domain.

Article Snippet: The normal human mammary epithelial cell line MCF10A (ATCC) was cultured in Mammary Epithelial Cell Growth Basal Medium, including hEGF (20 ng/ml), insulin (10 μg/ml), hydrocortisone (0.5 μg/ml) and bovine pituitary extract (50 μg/ml) (SingleQuots TM Kit; Lonza Group, Ltd.) containing streptomycin-penicillin (100 U/ml).

Techniques: MTS Assay, Flow Cytometry, Western Blot, Expressing, Control, Binding Assay, Activity Assay, Screening Assay, Labeling, Comparison, Staining, Microscopy, Immunofluorescence, Software, Fluorescence