Journal: Nature methods

Article Title: Simultaneous profiling of 3D genome structure and DNA methylation in single human cells

doi: 10.1038/s41592-019-0547-z

Figure Lengend Snippet: (a) tSNE of single-cell mC profiles of mouse ES cells and NMuMG cells . (b) Chromosome wide Pearson correlation matrix from pooled sc-m3C-seq maps for ES cells and NMuMG cells . (c-d) Principal component analysis (PCA) of whole genome contact matrices from sc-m3C-seq from ES from NMuMG cells (Percentage of variance are marked on the axis). PC1 and PC2 are shown in (c); PC1 and PC3 are shown in (d). (e) PCA of local interactions (<2Mb) from sc-m3C-seq data from NMuMG cells showing PC1 and PC2. (f) Correlation of PC1 and per cell contacts . For (a) and (c-f), n=2 independently prepared mouse ES cell cultures were analyzed. The two mESC replicates each contained 379 and 93 cells. One (n=1) replicate of NMuMG cells containing 96 cells was analyzed.

Article Snippet: NMuMg cells (RBRC-RCB2868) were obtained from the RIKEN BioResource Center.

Techniques:

Journal: bioRxiv

Article Title: Upregulated-flotillins and sphingosine kinase 2 derail vesicular trafic to stabilize AXL and promote epithelial-mesenchymal transition

doi: 10.1101/2020.05.12.090571

Figure Lengend Snippet: A) Lysates of NMuMG cells that stably express mCherry (NMuMGmCh) or both flotillin 1-HA and flotillin 2-mCherry (NMuMGF1F2) were analyzed by western blotting using antibodies against flotillin 1 and flotillin 2. In NMuMGF1F2 cells, flotillin levels were comparable to those detected in the invasive mammary tumor MDA-MB-231 cell line. Lysates from MDA-MB-231shFlot2 cells (generated using a shRNA against FLOT2 to knockdown both flotillins ) also were analyzed. Actin was used as loading control. B) Confocal images showing that endogenous flotillin 1 and 2 are localized at the plasma membrane in NMuMGmCh cells. In NMuMGF1F2 cells, they translocate to vesicular compartments, where they co-localize, as previously shown in MCF10AF1F2 and in MDA-MB-231 cells . C) Confocal images showing the co-localization of flotillin 1-HA and flotillin 2-mCherry in intracellular vesicles in NMUMGF1F2 cells. D, K) RT-qPCR analysis of the mRNA levels of E-cadherin, N-cadherin and vimentin (D) and of ZEB1 and ZEB2 (K) in NMuMGmCh and NMuMGF1F2 cells. Histograms show the mean ± SEM of 4 independent experiments. *P<0.05; **P<0.01. E, L) Western blotting performed using cell lysates from NMuMGmCh and NMuMGF1F2 cells to compare the amount of E- and N-cadherin (E), and ZEB1 (L). Histograms show the mean ± SEM of at least 4 independent experiments. *P<0.05; **P<0.01. F, G) Comparison of the distribution of endogenous E-, N-cadherin (F) and ZO-1 and vimentin (G) in NMuMGmCh and NMuMGF1F2 cells analyzed by immunofluorescence. Nuclei are stained with Hoechst (Hst, blue). H) Scanning electron microscopy images of the monolayer formed by NMuMGmCh and NMuMGF1F2 cells. I) Z-projection and 3D surface reconstruction of NMuMGmCh and NMuMGF1F2 cells after staining for F-actin, flotillin 2-mCherry and Hoechst (nuclei), performed with 35 plans and 22 plans every 0.3.µm for NMuMGmCh and NMuMGF1F2 respectively. J, K) Characterization of the transcriptional repressors of E-cadherin expression . RT-qPCR analysis of the mRNA levels of Snail1, Snail2, Twist in MCF10AmCh and MCF10AF1F2 cells (J) and ZEB1 and 2 in NMuMGmCh and NMuMGF1F2 cells (K). Histograms show the mean ± SEM of 3 independent experiments. M) Confocal images of ZEB1 localization and nuclei stained with Hoechst (Hst, blue) in NMuMGmCh and NMuMGF1F2 cells. Images inB, C, F, G, I and M are representative of cells observed in at least 3 independent experiments Scale bar: 10 µm.

Article Snippet: Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

Techniques: Stable Transfection, Western Blot, Generated, shRNA, Quantitative RT-PCR, Immunofluorescence, Staining, Electron Microscopy, Expressing

Journal: bioRxiv

Article Title: Upregulated-flotillins and sphingosine kinase 2 derail vesicular trafic to stabilize AXL and promote epithelial-mesenchymal transition

doi: 10.1101/2020.05.12.090571

Figure Lengend Snippet: A) GO identification of the biological process, cellular component and molecular function terms associated with the 802 genes that were differentially expressed in MCF10AF1F2 cells compared with MCF10AmCh cells. B) Venn diagrams comparing the genes that we found to be up- or down-regulated (> or <2.8, p<0.05) by RNA-seq data analysis in MCF10AF1F2 versus MCF10AmCh cells with the EMT signature established by . C) Plots from gene set enrichment analysis (GSEA) showing the enriched EMT-signature genes (up-regulated, upper panel; down-regulated, lower panel) in the ranked transcriptome profile. Green lines represent enrichment profiles. NES: normalized enrichment score. FDR: false discovery rate. Each hit from the EMT signature is represented by a vertical black bar, positioned on the ranked transcriptome profile with color-coded fold change values. D) List of the genes with the highest fold change among the genes up- and down-regulated and found in MCF10AF1F2 cells and found also in the EMT-transcriptomic signature P values were adjusted for multiple testing using the Benjamini-Hochberg procedure. D) List of the genes with the highest fold change among the genes up- and down-regulated and found in MCF10AF1F2 cells and found also in the EMT-transcriptomic signature P values were adjusted for multiple testing using the Benjamini-Hochberg procedure. E) RT-qPCR analysis of the mRNA levels E-cadherin, N-cadherin and vimentin in MCF10AmCh and MCF10AF1F2 cells. Data are the mean ± SEM of four independent experiments. *P<0.05. F) Western blot analysis of whole MCF10AmCh and MCF10AF1F2 cell lysates to investigate the expression of E- and N-cadherin, tubulin and flotillin 1. Data are the mean ± SEM of at least four independent experiments. *P<0.05; **P<0.01. G, H) Confocal images showing the distribution of E- and N-cadherin, flotillin 2-mCherry and ZO-1 in MCF10AmCh and MCF10AF1F2 cells and of vimentin in NMuMGmCh and NMuMGF1F2 cells, analyzed by immunocytochemistry. Nuclei were stained with Hoechst (Hst; blue). In G, for vimentin staining, the z projection corresponds to the stacked signal of 30 plans performed every 0.3 µm. I) RT-qPCR analysis of the mRNA levels of ZEB1 and ZEB2 in MCF10AmCh and MCF10AF1F2 cells. Histograms show the mean ± SEM of 4 independent experiments. *P<0.05. J) Western blotting to assess ZEB1, E-cadherin and α-tubulin expression in cell lysates from MCF10AmCh cells and MCF10AF1F2 cells transfected with siRNAs against ZEB1/2 or control siRNA against luciferase (CTL). Histograms (mean ± SEM of 4 independent experiments) show the quantification of the amount of each protein in the indicated conditions. K, L) Distribution of ZEB1 (K) and E-cadherin (L) in MCF10AmCh cells and in ZEB1/2 siRNA- or CTL siRNA-transfected MCF10AF1F2 cells analyzed by immunofluorescence using specific antibodies. In K, nuclei were stained with Hoechst (Hst). Images in G,H, K and L are representative of 3 independent experiments. Scale bar: 10 µm.

Article Snippet: Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Expressing, Immunocytochemistry, Staining, Transfection, Luciferase, Immunofluorescence

Journal: bioRxiv

Article Title: Upregulated-flotillins and sphingosine kinase 2 derail vesicular trafic to stabilize AXL and promote epithelial-mesenchymal transition

doi: 10.1101/2020.05.12.090571

Figure Lengend Snippet: A) Flotillin upregulation promotes AXL protein level increase . Cell lysates from MCF10AmCh and MCF10AF1F2 cells were probed by western blotting with antibodies against AXL phosphorylated on Y702, total AXL and tubulin. Results are expressed as fold-increase compared with MCF10AmCh cells and are the mean ± SEM of 6 independent experiments. B) Flotillin upregulation does not increase AXL mRNA level. RT-qPCR analysis of AXL expression in MCF10AmCh and MCF10AF1F2 cells. Results are expressed relative to the level in MCF10AmCh cells and are the mean ± SEM of 4 independent experiments. C) Flotillin upregulation stabilizes AXL. MCF10A and MCF10AF1F2 cells were incubated with cycloheximide (CHX, 100µg/ml) and cell lysates collected at the indicated time points. AXL and tubulin levels were assessed by western blotting (representative western blots are shown). The graph shows the normalized AXL levels in each cell line during CHX incubation. The results are the mean ± SEM of 6 to 8 independent experiments depending on the time point, and are expressed as the percentage of AXL level at T0. CHX treatment was stopped after 9 hours because MCF10Amch cells did not survive longer treatment. The difference is significant at time 6 hours ** ( p <0.01). D) AXL knock-down in MCF10AF1F2 cells decreases ZEB1 and N-cadherin expression . MCF10AmCh and MCF10AF1F2 cells were transfected with anti-luciferase (CTL) or AXL siRNAs and probed by western blotting with antibodies against the indicated proteins. The quantification results are the mean ± SEM of 4 independent experiments, and are expressed as fold-increase compared with control (MCF10AmCh). E) ZEB1 nuclear localization analyzed by immunocytochemistry and co-staining with Hoechst (Hst) and F) E-cadherin distribution analyzed by immunocytochemistry in MCF10AmCh and MCF10AF1F2 cells transfected with siRNA against luciferase (CTL) or AXL. Scale bar: 10 µm. F) Boyden chamber migration assay. MCF10AmCh and MCF10AF1F2 cells transfected with siRNA against luciferase (CTL) or AXL were seeded in the top of Boyden chamber inserts in serum-free medium, and serum-containing medium, acting as chemoattractant, was placed in the bottom well. Representative inverted-microscopy images of Hoechst-stained nuclei of cells that migrated through the pores. The histogram (mean ± SEM of at least four independent experiments) show the quantification of cell migration of the indicated cell lines. Scale bar: 50µm. Images in E, F, G are representative of at four independent experiments. * p <0.05, ** p <0.01,*** p <0.001

Article Snippet: Mouse antibodies used were against: actin (A5441, Sigma), flotillin 1 and flotillin 2 (BD Biosciences), E-cadherin (used for MCF10A cells, Life-technologies), E-cadherin (used for NMuMG cells, BD Biosciences), LAMP1 (used for MCF10A cells, BD Biosciences), Smad3 (ThermoFisher), vimentin and α-tubulin (Sigma), phosphorylated ERK1/2 at T202/Y204 and AKT (CST), pan-Ras (Millipore), phosphorylated Y (4G10), EphA4 (gift from Dr Greenberg, Harvard, Boston, USA) , and STAT3 (CST, #9139S).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Incubation, Transfection, Luciferase, Immunocytochemistry, Staining, Migration, Inverted Microscopy

Journal: FASEB BioAdvances

Article Title: Matrix Stiffness Regulates TGFβ1 ‐Induced αSMA Expression via a G9a‐ LATS ‐ YAP Signaling Cascade

doi: 10.1096/fba.2025-00117

Figure Lengend Snippet: Matrix stiffness regulates G9a and histone 3 lysine 9 dimethylation in response to TGFβ1. Immunofluorescence staining for (A) G9a and (B) H3K9me2 in NMuMG cells cultured on hydrogels and treated with or without TGFβ1. Scale bars: 25 μm. Quantification of the (C) relative nuclear G9a and (D) relative H3K9me2 levels from immunofluorescence images shown in panels (A) and (B). Data are normalized with respect to the soft hydrogel control sample. (E) Western blot for G9a using whole cell protein extracts and H3K9me2 using histone extracts from NMuMG cells cultured on hydrogels with and without TGFβ1 treatment. Relative quantification via densitometric analysis for (F) G9a and (G) H3K9me2 from blots shown in panel (E). Data are normalized with respect to the soft hydrogel control sample. All data represent mean ± sem for n = 3 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: NMuMG cells were authenticated by Genetica Cell Line Testing, a LabCorp brand, and tested negative for mycoplasma.

Techniques: Immunofluorescence, Staining, Cell Culture, Control, Western Blot, Quantitative Proteomics

Journal: FASEB BioAdvances

Article Title: Matrix Stiffness Regulates TGFβ1 ‐Induced αSMA Expression via a G9a‐ LATS ‐ YAP Signaling Cascade

doi: 10.1096/fba.2025-00117

Figure Lengend Snippet: Inhibition of G9a activity impacts H3K9 dimethylation levels and EMT in response to TGFβ1 and matrix stiffness. Immunofluorescence staining for (A) E‐cadherin and (B) αSMA for NMuMG cells cultured on hydrogels and treated with DMSO or the G9a inhibitor UNC0642 (10 nM) with and without TGFβ1 treatment. Scale bars: 25 μm. (C) Quantification of αSMA positive NMuMG cells for various treatment conditions from immunofluorescence staining for αSMA. Data represent mean ± sem for n = 4 independent experiments, ** p < 0.01, *** p < 0.001. (D) Western blots for H3K9me2, G9a, E‐cadherin, N‐cadherin, and αSMA in NMuMG cells. Densitometric quantification of the relative expression of (E) H3K9me2, (F) G9a, (G) E‐cadherin, (H) αSMA, and (I) N‐cadherin from western blots shown in panel (D). Data are normalized with respect to the soft hydrogel DMSO control sample. Data represent mean ± sem for n = 3 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: NMuMG cells were authenticated by Genetica Cell Line Testing, a LabCorp brand, and tested negative for mycoplasma.

Techniques: Inhibition, Activity Assay, Immunofluorescence, Staining, Cell Culture, Western Blot, Expressing, Control

Journal: FASEB BioAdvances

Article Title: Matrix Stiffness Regulates TGFβ1 ‐Induced αSMA Expression via a G9a‐ LATS ‐ YAP Signaling Cascade

doi: 10.1096/fba.2025-00117

Figure Lengend Snippet: siRNA knockdown of G9a attenuates TGFβ1‐induced changes in H3K9me2, αSMA, and N‐cadherin levels as a function of matrix stiffness. Immunofluorescence staining for (A) E‐cadherin and (B) αSMA in NMuMG cells transfected with NTC siRNA or siG9a#2 with and without TGFβ1 treatment. Scale bars: 25 μm. (C) Quantification of the percentage of αSMA positive cells for various treatment conditions. Data represent mean ± sem for n = 4 independent experiments, *** p < 0.001. (D) Western blot for H3K9me2, G9a, E‐cadherin, N‐cadherin, and αSMA in NMuMG cells transfected with NTC siRNA or siG9a#2 with and without TGFβ1 treatment. Densitometric quantification of the relative levels of (E) H3K9me2, (F) G9a, (G) E‐cadherin, (H) αSMA, and (I) N‐cadherin from blots shown in panel (D). Data are normalized with respect to the soft NTC siRNA control sample. Data represent mean ± sem for n = 3 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: NMuMG cells were authenticated by Genetica Cell Line Testing, a LabCorp brand, and tested negative for mycoplasma.

Techniques: Knockdown, Immunofluorescence, Staining, Transfection, Western Blot, Control

Journal: FASEB BioAdvances

Article Title: Matrix Stiffness Regulates TGFβ1 ‐Induced αSMA Expression via a G9a‐ LATS ‐ YAP Signaling Cascade

doi: 10.1096/fba.2025-00117

Figure Lengend Snippet: Knockdown of G9a impacts YAP subcellular localization and inhibiting YAP attenuates TGFβ1‐induced αSMA expression and cell morphology changes as a function of matrix stiffness. (A) Immunofluorescence staining for YAP in NMuMG cells transfected with NTC siRNA or siG9a#2 with and without TGFβ1 treatment. Scale bars: 25 μm. (B) Quantification of the percentage of cells with nuclear (N), pancellular (N/C), or cytoplasmic (C) YAP localization under different treatment conditions. Data represent mean ± sem for n = 3; # p < 0.001 with respect to all other samples, * p < 0.05 with respect to the stiff hydrogel siG9a control and soft hydrogel siG9a TGFβ1 samples. (C) Immunofluorescence staining for αSMA in NMuMG cells cultured on hydrogels and treated with DMSO or YAP inhibitor Verteporfin (4 μM) with and without TGFβ1 treatment. Scale bars: 25 μm. (D) Quantification of αSMA positive NMuMG cells cultured on soft and stiff hydrogels and treated with DMSO or Verteporfin in the presence and absence of TGFβ1. Data represent mean ± sem for n = 3 independent experiments; *** p < 0.001.

Article Snippet: NMuMG cells were authenticated by Genetica Cell Line Testing, a LabCorp brand, and tested negative for mycoplasma.

Techniques: Knockdown, Expressing, Immunofluorescence, Staining, Transfection, Control, Cell Culture

Journal: FASEB BioAdvances

Article Title: Matrix Stiffness Regulates TGFβ1 ‐Induced αSMA Expression via a G9a‐ LATS ‐ YAP Signaling Cascade

doi: 10.1096/fba.2025-00117

Figure Lengend Snippet: G9a regulates LATS kinase which acts upstream of YAP and αSMA. (A) Quantitative real‐time PCR for LATS2 and (B) relative levels of LATS2 quantified from immunofluorescence staining in NMuMG cells cultured on hydrogels with storage moduli of 260 Pa and 2200 Pa and transfected with non‐targeting control siRNA (NTC) or siRNA targeting G9a (siG9a#2) with and without TGFβ1 treatment. Data represent mean ± sem for n = 3 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) Quantification of the percentage of cells with nuclear (N), pancellular (N/C), or cytoplasmic (C) YAP localization in cells following treatment with DMSO or LATS1/2 kinase inhibitor TRULI (15 μM) with and without TGFβ1 treatment. Data represent mean ± sem for n = 3 independent experiments; * p < 0.05, ** p < 0.01 with respect to soft DMSO control sample, # p < 0.01 with respect to stiff DMSO control sample, a p < 0.001 with respect to all other samples except stiff TRULI TGFβ1, b p < 0.001 with respect to all other samples. Immunofluorescence staining for (D) YAP and (E) αSMA in NMuMG cells under different treatment conditions. Scale bars: 25 μm. (F) Quantification of αSMA positive NMuMG cells treated with DMSO or TRULI in the presence and absence of TGFβ1. Data represent mean ± sem for n = 3 independent experiments; ** p < 0.01, *** p < 0.001. (G) Western blot for E‐cadherin and αSMA in NMuMG cells cultured on hydrogels and treated with DMSO or TRULI with and without treatment with TGFβ1. Densitometric quantification of the relative expression of (H) E‐cadherin and (I) αSMA from western blots shown in panel (G). Data are normalized with respect to the soft DMSO control sample. Data represent mean ± sem for n = 3 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: NMuMG cells were authenticated by Genetica Cell Line Testing, a LabCorp brand, and tested negative for mycoplasma.

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Cell Culture, Transfection, Control, Western Blot, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

doi: 10.3390/ani11061548

Figure Lengend Snippet: Comparison of Saa1 and Saa3 mRNA expression levels in NMuMG cells treated with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Data are presented as the mean ± standard deviation of four independent experiments. Asterisks indicate significant difference compared with control levels: ** p < 0.01. NMuMG, normal murine mammary gland.

Article Snippet: Normal murine mammary gland (NMuMG) epithelial cells were purchased from the European Collection of Authenticated Cell Cultures General Cell Collection, Public Health England (94081121, Salisbury, UK).

Techniques: Comparison, Expressing, Standard Deviation, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

doi: 10.3390/ani11061548

Figure Lengend Snippet: Comparison of SAA1 (A and B) and SAA3 (C and D) protein expression levels in NMuMG cells treated with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by immunofluorescence analysis (IFA). ( A , C ) Fields of view where fluorescence intensity was measured. Scale bar = 20 µm. ( B , D ) The relative SAA1 and SAA3 protein expression levels in NMuMG cells following stimulation with LPS or LTA were normalized to those in untreated, control cells. Data are presented as the mean fluorescence of five or more random locations with vertical bars representing standard deviation. Asterisks indicate significant difference compared with control levels: * p < 0.05, ** p < 0.01. NMuMG, normal murine mammary gland.

Article Snippet: Normal murine mammary gland (NMuMG) epithelial cells were purchased from the European Collection of Authenticated Cell Cultures General Cell Collection, Public Health England (94081121, Salisbury, UK).

Techniques: Comparison, Expressing, Immunofluorescence, Fluorescence, Control, Standard Deviation

Journal: Animals : an Open Access Journal from MDPI

Article Title: Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

doi: 10.3390/ani11061548

Figure Lengend Snippet: Detection of SAA1 and SAA3 protein expression in NMuMG cell supernatants treated with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by Western blot. ( A ) Detection of SAA1 in the cell supernatant. Membranes were incubated with the primary anti-mouse SAA1 antibody (dilution of 1:500, 60 s exposure time). ( B ) Detection of SAA3 in the cell supernatant. Membranes were incubated with the primary anti-mouse SAA3 antibody (dilution of 1:50, 120 s exposure time). PC, positive control; 1, recombinant murine SAA1 (rSAA1); 3, rSAA3; NMuMG, normal murine mammary gland.

Article Snippet: Normal murine mammary gland (NMuMG) epithelial cells were purchased from the European Collection of Authenticated Cell Cultures General Cell Collection, Public Health England (94081121, Salisbury, UK).

Techniques: Expressing, Western Blot, Incubation, Positive Control, Recombinant