human breast epithelial cells mcf10a  (Procell Inc)

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: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: siSPRR3 depletion results in a reduction in the number of nucleoli per nucleus in MCF10A cells. A , siSPRR3 depletion with siGENOME siRNAs reduces the number of nucleoli per nucleus. Images and histograms from a genome-wide screen using Dharmacon/Horizon siGENOME siRNAs . The images show nuclei (Hoechst, blue ) and nucleoli (anti-fibrillarin, red ) after 72 h of treatment with the negative control (siGFP), positive control (siUTP4), or siSPRR3 siRNAs. Histograms show the distribution of cells that have the indicated number of nucleoli per nucleus. Light grey shows the distribution for the negative control, black shows the test condition, and dark grey shows overlap of the two frequencies. B , siSPRR3 depletion with siON-TARGET (siONT) SMARTPool siRNA reduces the number of nucleoli per nucleus. The images show nuclei and nucleoli after 72 h of treatment with the negative control (non-targeting siRNA, siNT), positive control (siUTP4), or siSPRR3 siRNAs. The shading in the histograms is as in A ). The data were collected across four replicates which are pooled in the histogram. C , siSPRR3 depletion with individual siONT SMARTPool siRNAs (deconvolution) reduces the number of nucleoli per nucleus. The images show nuclei and nucleoli after 72 h of treatment with the negative control (siNT), positive control (siNOL11), or representative siSPRR3 individual siRNAs (SPRR3-si1 and SPRR3-si2). The shading in the histograms is as in ( A ). The histograms include all three replicates pooled. The table summarizes each of four individual siONT SMARTPool siRNAs, showing that a reduction in nucleolar number correlates with a loss in cell viability. D , siSPRR3 (custom subpool of SPRR3-si1 and SPRR3-si2) reduces SPRR3 mRNA levels in MCF10A cells. RT-qPCR data of SPRR3 mRNA demonstrating knockdown after 72 h. The data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. The mean ± SEM are shown alongside individual data points, colored by replicate. E , siSPRR3 (custom subpool of SPRR3-si1 and SPRR3-si2) reduces SPRR3 protein levels in MCF10A cells. Western blot of SPRR3 protein demonstrating decreased levels after 72 h. Protein levels were normalized to total protein (trichloroethanol total protein stain), then to siNT. The mean ± SEM are shown alongside individual data points, colored by replicate. This sample was run on the same Western blot as in . After imaging total protein on the membrane, the blot was cut between 10 to 15 kD markers to stain separately for SPRR3 ( E , above) or RPS28 . Data in ( D and E ) were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. ∗, p < 0.05; ∗∗∗, p < 0.001.

Article Snippet: Human MCF10A breast epithelial cells (#CRL-10317, American Type Culture Collection) were cultured in DMEM/nutrient mixture F-12 (Gibco 11330032) with 5% horse serum (Gibco 16050122), 20 ng/ml epidermal growth factor (Peprotech AF1005), 0.5 μg/ml hydrocortisone (MilliporeSigma H0135), 100 ng/ml cholera toxin (MilliporeSigma C8052), and 10 μg/ml insulin (MilliporeSigma I1882).

Techniques: Genome Wide, Negative Control, Positive Control, Quantitative RT-PCR, Knockdown, Comparison, Western Blot, Staining, Imaging, Membrane

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 depletion reduces pre-rRNA transcription. A , schematic of the major steps in ribosome biogenesis in human cells. B , SPRR3 depletion inhibits nucleolar rRNA biogenesis. Representative images and quantification of control or SPRR3-depleted MCF10A cells (siONT SMARTpool) following anti-fibrillarin (FBL) staining and 5-EU incorporation. Scale bars are 10 μm. siNT is a non-targeting negative control siRNA. siPOLR1A is a positive control targeting the RNAPI subunit, POLR1A. The overall mean percent inhibition ± SEM is shown for each treatment, with each dot representing one well. Each well in the siSPRR3 condition represents a separate day of testing and control datapoints are distributed across the three testing days. 0% inhibition is determined by the mean value of siNT, and 100% inhibition is set to the mean value of the siPOLR1A positive control. C , RT-qPCR analysis shows decreased 47S/45S pre-rRNA levels upon SPRR3 depletion in MCF10A cells. The mean ± SEM are shown alongside individual data points, colored by replicate. The data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. D , dual-luciferase reporter assay shows RNAPI promoter activity is reduced after SPRR3 depletion. The mean ± SEM are shown alongside individual data points, colored by replicate. The controls in these data have also been published in , without the inclusion of siSPRR3. All the data in this figure were analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Human MCF10A breast epithelial cells (#CRL-10317, American Type Culture Collection) were cultured in DMEM/nutrient mixture F-12 (Gibco 11330032) with 5% horse serum (Gibco 16050122), 20 ng/ml epidermal growth factor (Peprotech AF1005), 0.5 μg/ml hydrocortisone (MilliporeSigma H0135), 100 ng/ml cholera toxin (MilliporeSigma C8052), and 10 μg/ml insulin (MilliporeSigma I1882).

Techniques: Control, Staining, Negative Control, Positive Control, Inhibition, Quantitative RT-PCR, Comparison, Luciferase, Reporter Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 depletion causes reduced global translation (protein synthesis). A , representative image and quantification of puromycin incorporation shows reduced translation after SPRR3 depletion in MCF10A cells. α-puromycin shows puromycin incorporation as a proxy for global protein synthesis. Total protein is the trichloroethanol total protein stain loading control. Images were quantified with Bio-Rad Image Lab. The mean ± SEM are shown alongside individual data points, colored by replicate. siRPL4 is the positive control. Data were normalized to a non-targeting siRNA (siNT), then graphed and analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗∗∗, p < 0.001. The control data in this puromycin Western blot have also been published in without the SPRR3 data. B , summary table describing the effects of SPRR3 depletion on ribosome biogenesis and the nucleolus. SPRR3 depletion reduces nucleolar number, nucleolar rRNA biogenesis (5-EU incorporation assay), pre-rRNA transcript levels (RT-qPCR of 47S/45S rRNA), rDNA promoter activity (luciferase reporter assay), and global protein synthesis (puromycin incorporation assay). SPRR3 depletion was found to have no effect on the ratio of 18S to 28S rRNA nor to produce changes in pre-rRNA northern blots, indicating that SPRR3 does not affect rRNA processing. Taken together, this suggests that SPRR3 plays a role in pre-rRNA transcription and nucleolar rRNA biogenesis that is essential to the normal translational activity of ribosomes.

Article Snippet: Human MCF10A breast epithelial cells (#CRL-10317, American Type Culture Collection) were cultured in DMEM/nutrient mixture F-12 (Gibco 11330032) with 5% horse serum (Gibco 16050122), 20 ng/ml epidermal growth factor (Peprotech AF1005), 0.5 μg/ml hydrocortisone (MilliporeSigma H0135), 100 ng/ml cholera toxin (MilliporeSigma C8052), and 10 μg/ml insulin (MilliporeSigma I1882).

Techniques: Staining, Control, Positive Control, Western Blot, Quantitative RT-PCR, Activity Assay, Luciferase, Reporter Assay, Northern Blot

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 depletion causes the nucleolar stress response in MCF10A and A549 cells. A , schematic of the nucleolar stress response in human cells. Disruption of ribosome biogenesis causes accumulation of free 5S RNP which inhibits the ubiquitin ligase MDM2, leading to accumulation of TP53 and increased transcription of CDKN1A . B , TP53 stabilization after SPRR3 depletion in MCF10A cells. Representative images and quantification of TP53 Western blots after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. TP53 levels were normalized to total protein (trichloroethanol stain), then siNT. C , CDKN1A mRNA levels are elevated after SPRR3 depletion in MCF10A cells. After 72 h of siSPRR3 treatment, CDKN1A mRNA transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. These data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. D , siSPRR3 reduces SPRR3 mRNA levels in A549 cells. After 72 h of siSPRR3 treatment, SPRR3 transcript levels were detected by RT-qPCR. The data were normalized as in ( C ). E , siSPRR3 reduces SPRR3 protein levels in A549 cells. After 72 h of treatment with siSPRR3, SPRR3 protein levels were detected by Western blot. SPRR3 levels were normalized as in ( B ). F , SPRR3 depletion lowers 47S/45S pre-rRNA levels in A549 cells. Primers to the 5′ ETS of the 47S/45S rRNA were used to detect pre-rRNA levels after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. The data were normalized as in ( C and D ). G , TP53 stabilization after SPRR3 knockdown in A549 cells. Representative images and quantification of TP53 Western blots after 48h or 72h of treatment with siSPRR3 are shown. Protein levels were normalized as in ( B and E ). H , CDKN1A levels are elevated after SPRR3 depletion in A549 cells. After 72 h of treatment with siSPRR3, CDKN1A transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. Data were normalized as in ( C ). The mean ± SEM are shown alongside individual data points. For graphs with two conditions, data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. For graphs with more than two conditions, data were analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Human MCF10A breast epithelial cells (#CRL-10317, American Type Culture Collection) were cultured in DMEM/nutrient mixture F-12 (Gibco 11330032) with 5% horse serum (Gibco 16050122), 20 ng/ml epidermal growth factor (Peprotech AF1005), 0.5 μg/ml hydrocortisone (MilliporeSigma H0135), 100 ng/ml cholera toxin (MilliporeSigma C8052), and 10 μg/ml insulin (MilliporeSigma I1882).

Techniques: Disruption, Ubiquitin Proteomics, Western Blot, Positive Control, Staining, Quantitative RT-PCR, Comparison, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 drives AKT phosphorylation and maintains POLR1A levels. A , AKT phosphorylation at serine 473 (pAKT) is decreased after a 72h SPRR3 depletion in MCF10A cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). Blots were probed for pAKT, then stripped and re-probed for total AKT. Signal was measured in Bio-Rad Image Lab. pAKT levels were normalized to total protein and total AKT. Total AKT was normalized to total protein, ensuring no significant difference in overall AKT levels upon SPRR3 depletion. B , phosphorylated AKT (pAKT) levels are decreased after 72h SPRR3 depletion in A549 cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). C , POLR1A levels are decreased upon 72h SPRR3 depletion. Representative image of Western blotting and quantification of POLR1A levels in MCF10A cells. siPOLR1A was used as a positive control. D , summary of effects of siSPRR3 depletion that we have confirmed in both MCF10A cells and A549 cells. For all graphs in this figure, the mean ± SEM are shown alongside individual data points. Data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns, not significant.

Article Snippet: Human MCF10A breast epithelial cells (#CRL-10317, American Type Culture Collection) were cultured in DMEM/nutrient mixture F-12 (Gibco 11330032) with 5% horse serum (Gibco 16050122), 20 ng/ml epidermal growth factor (Peprotech AF1005), 0.5 μg/ml hydrocortisone (MilliporeSigma H0135), 100 ng/ml cholera toxin (MilliporeSigma C8052), and 10 μg/ml insulin (MilliporeSigma I1882).

Techniques: Phospho-proteomics, Staining, Western Blot, Positive 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 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