nmumg cells  (Genetica Inc)

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a . Schematic drawing of the transwell-platform. Serum deprived NMuMG cells were covered with an agarose disk followed by a metal load of defined weight, exerting uniaxial compressive force. At the indicated times, the load was removed, and the cells were harvested for different assays. b . NMuMG cells were exposed (+) or not (-) to compression and allowed to migrate for 24 hours through 8 µm transwell pores. Transmigrated cells adhering to the bottom of the 6-well plate form colonies, which were stained with crystal violet. The colony-forming units were quantified to compare the migratory potential across conditions. N=18; unpaired t test comparison; p<0.0001****; p<0.05 considered significant. c . and d . Serum deprived NMuMG cells invading the empty space were imaged by phalloidin staining 24 hours after removing the inset of the ibidi chambers, in the presence (+) or absence (-) of compressive pressure. Scale bar: 50 μm ( c ). Note that compressed cells acquire an elongated, migratory-like phenotype, due to a profound rearrangement of their cytoskeleton. The roughness of the edge was quantified ( d ) as described in . The normalized wound edge perimeter was plotted as a metric of the compressive stress phenotype. Unpaired t-test comparison; p<0.0001****; p<0.05 considered significant. e , f and g . NMuMG cells under normal (-) or compressive stress (+) conditions were stained with DAPI (grey and blue) or rhodamine-phalloidin (grey and green), to visualize nuclei or the actin cytoskeleton, respectively ( e ). Scale bar: 20 μm. The inset magnifies a section of cells at the epithelia edge. Scale bar: 3 μm. The nuclear ( f ) and cell ( g ) shape changes were quantified as an increase in the cell or nuclear aspect ratio after cellpose-based cell or nuclear segmentation. N=11 and N=8, respectively; Unpaired t-test comparison; p<0.0001****; p<0.05, considered significant. h . The adherens junction marker E-cadherin (gray and green) and the tight junction protein ZO1 (gray and magenta) were immunostained in compressed (+) and non-compressed (-) NMuMG cells. Scale bar: 20 μm. The inset magnifies a section of cells at the epithelia edge. Note that upon compression, E-cadherin is internalized and forms punctate structures, while ZO1 disappears from tight junctions.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Staining, Comparison, Marker

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. Experimental outline of compressive stress application on the transwell platform. b. NMuMG cells were exposed (+) or not (-) to compression and allowed to migrate for 24 hours through narrow 8 µm transwell pores. Transmigrated cells adhere to the bottom of the 6-well plate form colonies, which were stained with crystal violet. Representative images of colonies are shown. c. Wound edge roughness quantification. Images of the tissue edges obtained with ibidi chambers are converted to a mask image, and the perimeter of the edge is divided by a straight line connecting two edge points. d. Schematic workflow to quantify the nuclear and cell morphological changes.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Staining

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. Schematic representation of the ERK5 MAPK pathway. ERK5 is phosphorylated and thereby activated by MEK5 on the TY residues in its T-loop. Active ERK5 phosphorylates cytoplasmic targets, and translocates into the nucleus, where it binds and phosphorylates the transcription factor MEF2. In the reporter construct, active MEF2 drives expression of GFP ( MEF2p -GFP). b. NMuMG cells were reverse-transfected with the MEF2p -GFP reporter plasmid and either an empty control vector (EV) or a vector expressing an HA-tagged dominant-active MEK5 mutant (HA-MEK5DD). GFP expression was monitored with (+) or without (-) compressive stress after 24 hours by western blot analysis. Where indicated (+), XMD8-92 was added to inhibit ERK5 activity. α-tubulin is used as a loading control. The numbers below represent densitometric quantification of GFP level across conditions, normalized to the tubulin loading control and relative to the empty control vector in the absence of compression and ERK5 inhibition. c. Western blot (WB) analysis confirmed elevated levels of active ERK5 (p-ERK5) after 3 hours of compression in serum-starved NMuMG cells, while the total ERK5 levels remained unchanged. α-tubulin controls equal loading. The numbers below represent the densitometric quantification of p-ERK5 and ERK5 levels across conditions normalized to GAPDH levels and relative to control condition without compression and ERK5 depletion. d - f. NMuMG cells siRNA-depleted for ERK5 (siERK5) or treated with a control siRNA oligo (siCTR) were exposed (+) or not (-) to compressive stress for 3 hours and the localization of p-ERK5 was analyzed by immunofluorescence analysis. Representative images show increased p-ERK5 at cell-cell contacts ( d ). Scale bar: 20 μm. Total- ( e ) and epithelial ( f ) p-ERK5 fluorescence intensity was quantified, and statistical significance confirmed by an ANOVA followed by Tukey multiple comparison test; ***p<0.001; p<0.05 was considered statistically significant.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Construct, Expressing, Transfection, Plasmid Preparation, Control, Mutagenesis, Western Blot, Activity Assay, Inhibition, Immunofluorescence, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. NMuMG cells siRNA depleted for ERK5 (siERK5) or treated with a control oligo (siCTR) were exposed (+) or not (-) to physical compression and allowed to migrate for 24 hours through 8 μm transwell pores. Colonies formed by transmigrated cells were visualized by crystal violet staining and quantified as described in . N=6; ANOVA followed by Tukey multiple comparison test; ***p<0.001; p<0.05, considered statistically significant. b - d. Invading NMuMG cells growing in ibidi chambers, either siRNA-depleted for ERK5 or treated with control oligos ( b, c ) or exposed to the ERK5 inhibitor XMD8-92 or DMSO solvent control ( d ), were visualized by phalloidin staining in the presence (+) or absence (-) of compressive pressure for 24 hours. The wound edge roughness was quantified to compare the different conditions. N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. e. and f. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector expressing dominant-active HA-MEK5DD. Where indicated, the ERK5 inhibitor XMD8-92 or solvent control (DMSO) was added. The wound edge roughness was visualized by phalloidin staining ( e ) and quantified to compare the different conditions ( f ). N=3; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. g - i. NMuMG cells siRNA-depleted for ERK5 (siERK5) or treated with control oligos (siCTR) exposed (+) or not (-) to compressive force conditions were stained with DAPI, rhodamine-phalloidin, and immunostained with antibodies against ZO1 to visualize tight junctions. Scale bar: 20 μm. The cell shape changes ( h ) and nuclear aspect ratio ( i ) were quantified as described in the legend to and , respectively. N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Control, Staining, Comparison, Solvent, Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. and b . crystal violet staining of transmigrated NMuMG colonies exposed (+) or not (-) to compression and treated with siRNA controls (siCTR) or oligos targeting ERK5 (siERK5) ( a ), or upon ERK5 inhibition with XMD8-92 or the DMSO-solvent control ( b ). c. Quantification of colony forming units (CFU) of b . N=8; Statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test; ***p<0.001; p<0.05, considered significant. d. Invading NMuMG cells growing in ibidi chambers treated with XMD8-92 or for control with DMSO, were exposed (+) or not (-) to compression. After 24 hours, the cells were stained with phalloidin and filamentous actin visualized by microscopy. Scale bar: 50 μm. e. Representative images of NMuMG cells with (+) or without (-) compression were stained with DAPI to visualize the nuclei and phalloidin to probe filamentous actin. As indicated, the cells were treated with XMD8-92 or DMSO for control. Scale bar: 20 μm. f. Cell shape changes were quantified as the increase in the cell aspect ratio after Cellpose-based cell segmentation. N=4; ANOVA and Tukey’s multiple comparison; ***p<0.001; p<0.05, considered statistically significant. g. Morphological changes of nuclei are quantified as the increase in nuclear aspect ratio after Cellpose-based nuclear segmentation. N=4; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. Note that upon compression, ERK5 activity is required for actin rearrangement and cell-cell contact dissolution.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Staining, Inhibition, Solvent, Control, Comparison, Microscopy, Activity Assay, Dissolution

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a – c. NMuMG cells exposed (+) or not (-) to compression were treated with XMD8-92 or for control with DMSO ( a , c ), or with siRNA controls (siCTR) or sioligos targeting ERK5 (siERK5) ( b ). Cells were then stained with phalloidin to visualize filamentous actin and antibodies against E-cadherin ( a , b ) or ZO1 ( c ). Scale bar: 20 μm. ERK5 activity is required for compression-induced dissolution of tight and adherens junctions as well as E-cadherin internalization. d. Confocal images of actin and ZO1 in NMuMG cells transfected with a control vector or a plasmid overexpressing HA-MEK5DD to constitutively activate ERK5. As indicated the cells were treated with the ERK5 inhibitor XMD8-92 or for control DMSO. Scale bar: 20 μm. Note that HA-MEK5DD overexpression promotes an invasive morphology even in the absence of compression.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Control, Staining, Activity Assay, Dissolution, Transfection, Plasmid Preparation, Over Expression

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. Experimental workflow used for phospho-proteomic profiling of NMuMG cells to classify compression-regulated, ERK5-dependent and independent phospho-sites. Phospho-peptides were identified by MS-analysis after F e -NTA enrichment, using extracts prepared from serum-starved NMuMG cells exposed (filled circles) or not (open circles) to compression for 3 hours, with or without ERK5 inhibition by XMD8-92. b. - e. Volcano plot showing differential protein phosphorylation in compression versus no-compression comparison (p-adj. ≤ 0.05, log2FC>1.0) marking ERK5-independent ( b ) and ERK5-dependent ( c ) compression-induced phosphosites. Proteins involved in cell-to-cell contact and the regulation of the actin cytoskeleton ( c ) and gene expression regulation and nuclear import ( b ) are highlighted, respectively. Each phosphorylated protein is represented by its most significant phosphopeptide. The p-value <0.05 is considered significant and corresponds to an ANOVA two-sided p-value, adjusted for multiple testing across the cells-treatment conditions of interest after post hoc analysis (FDR). Gene ontology (GO) enrichment analysis revealed ERK5-dependent targets related to cell-cell junction and actin cytoskeleton organization ( e ), and pressure-induced, but ERK5-independent targets related to nuclear transport regulation and post-transcriptional gene silencing ( d ). Bonferroni- adjusted p-value < 0.05 corresponds to two-sided Fisher’s exact test. All genes in Mus musculus database are used as reference list. f. Experimental workflow to functionally validate candidate ERK5-substrates. Candidates were siRNA depleted in serum starved NMuMG cells growing in ibidi chambers and examined by the epithelial edge assay after 24 hours of compression. The screen was repeated twice, each time dividing the candidates into five or six batches, with each batch including non-targeting siRNAs and siERK5 controls. Cells were fixed and stained with rhodamine phalloidin and the edge roughness imaged and quantified. The resulting phenotypes were grouped in 4 functional categories, which are illustrated by the edge images of NMuMG cells exposed (+) or not (-) to compressive pressure g . Category 1: siRNA knockdown has no influence on the phenotypic response to compression; category 2: siRNA knockdown prevents the response to compression; category 3: siRNA knockdown increases cell dissociation upon compression; category 4: siRNA knockdown leads to cell dissociation even in the absence of compression. h. and i. Graphic comparison of wound edge roughness of two screen replicates. The wound edge roughness of each target is normalized to the average value of the control siRNAs and log2 transformed. The dashed lines indicate 95% confidence intervals (95% CI) around the median of all measurements in each independent screen. Candidates that significantly reduce the wound edge perimeter upon compression compared to cells treated with control siRNAs are marked in blue. Candidates enhancing the wound edge roughness with ( i ) or without compression ( h ) are marked in red and green, respectively.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Inhibition, Phospho-proteomics, Comparison, Gene Expression, Staining, Functional Assay, Knockdown, Control, Transformation Assay

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. Schematic illustration of the ERK5 signaling pathway phosphorylating S909 of the myosin light chain phosphatase subunit MYPT1, which in turn dephosphorylates and thereby activates the myosin light chain (MLC). Active myosin controls cortical tension by pulling on actin fibers. b . and c . NMuMG cells siRNA depleted for ERK5 (siERK5), MYPT1 (siMYPT1) or treated with a control oligo (siCTR) were exposed (+) or not (-) to physical compression for 3 hours, and immunostained for phospho-MLC (p-MLC). Note that MLC phosphorylation is decreased upon compression by an ERK5 and MYPT1-dependent mechanism. Scale bar: 20 μm ( b ). Fluorescence intensity of p-MLC at cell edges was quantified as described for phospho-ERK5. Compression significantly reduces epithelial p-MLC levels while ERK5 depletion protects p-MLC at cell-cell contacts ( c ). d. Extracts prepared from cells expressing HA-tagged ERK5 (HA-ERK5) or carrying an empty control plasmid (HA-empty) were incubated with HA-affinity resins. Immobilized proteins were incubated with a peptide encompassing the S909 phosphorylation site of MYPT1 (S909-WT) or a mutant peptide where Ser909 was replaced by an alanine residue (S909-A) in the presence of γ- 32 P-ATP. The peptides were separated by thin layer chromatography (TLC) and analyzed by autoradiography. The numbers below represent the densitometric quantification of phosphorylated peptides relative to active ERK5 upon compression. e. - g. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector overexpressing wild-type MYPT1 (Ser909-WT), or MYPT1 mutants changing S909 either to a phosphomimicking glutamic acid (Ser909-D) or a non-phosphorylatable alanine (Ser909-A) residue. Wound edge roughness was visualized by phalloidin staining ( e ) and quantified to compare the different conditions ( f ). N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. Note the elongated morphology and significantly weaker intercellular connections in NMuMG cells expressing Ser909-D compared to Ser909-WT and Ser909-A mutants ( g ).

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Control, Phospho-proteomics, Fluorescence, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Residue, Thin Layer Chromatography, Autoradiography, Transfection, Staining, Comparison

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a . – c : NMuMG cells exposed (+) or not (-) to compression were treated with siRNA controls (siCTR) or sioligos targeting ERK5 (siERK5) or MYPT1 (siMYPT1). Cells were stained for total MLC ( a ), non-muscle myosin IIA heavy chain ( b ), or the cell tension marker vinculin ( c ), and visualized by immunofluorescence. Scale bar: 20 µm. d. Quantification Ser909 phosphorylation of MYPT1 using PRM analysis. NMuMG cells were compressed in the presence or absence of XMD8-92 (inhibitor). N=3; One-tailed t-test; ***p<0.001; p<0.05, considered statistically significant. e. Analysis of total MYPT1 levels in NMuMG cells compressed in the presence or absence of XMD8-92 (inhibitor) using phosphoproteomic analysis. N=3; One-tailed t-test; ***p<0.001; p<0.05, considered statistically significant. f. HA-tagged ERK5 was immobilized on HA-beads and incubated in the presence of γ 32 P-ATP with a peptide encompassing the Ser909 phosphorylation site of MYPT1, either as wild-type (S909-WT) or the S909 alanine mutant (S909-A). As indicated, the in vitro kinase assay was performed in the presence of XMD8-92 or DMSO for control. g. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector overexpressing myc-tagged wild-type MYPT1 (Ser909-WT), or MYPT1 mutants changing S909 either to a phosphomimicking glutamic acid (Ser909-D) or a non-phosphorylatable alanine (Ser909-A) residue. Expression levels were visualized by immunoblotting with myc-antibody (upper blot). An antibody against tubulin (Tub) controls equal loading. While Ser909-A is overexpressed, Ser909-WT and Ser909-D are present at comparable levels.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Staining, Marker, Immunofluorescence, Phospho-proteomics, One-tailed Test, Incubation, Mutagenesis, In Vitro, Kinase Assay, Control, Transfection, Plasmid Preparation, Residue, Expressing, Western Blot

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. NMuMG cells siRNA depleted for ROCK (siROCK), ERK5 (siERK5), or both together (siROCK + siERK5), or treated with a control oligo (siCTR), were exposed (+) or not (-) to physical compression for 3 hours, and phospho-threonine 852 (T852) of MYPT1 (p-MYPT1) was analyzed by immunofluorescent staining. Scale bar: 20 μm. Note that the ROCK-dependent T852 phosphorylation is decreased upon compression. b. and c. NMuMG cells grown in ibidi chambers were treated with the ROCK inhibitor Y27632, its solvent DMSO, or together with the ERK5 inhibitor XMD8-92. Cells were exposed (+) or not (-) to compressive pressure for 24 hours, and wound edge roughness was quantified after phalloidin staining. Note that ROCK and ERK5 activities antagonize each other. N=5; ANOVA followed by post hoc Bonferroni multiple comparison of relevant conditions; ***p<0.001; p<0.05 considered statistically significant. d . During unstressed conditions, ROCK phosphorylates MYPT1 on its inhibitory Thr852 site (Thr853 in human), thereby increasing active myosin (phospho-MLC) and stabilizing actin structures and cell-cell junctions. Compressive stress inhibits ROCK but activates ERK5, which then phosphorylates MYPT1 on Ser909. This phosphorylation switch activates the myosin light chain phosphatase complex (MLCP), leading to myosin inhibition and in turn changes in cortical actin structures and disruption of cell-cell junctions, thus promoting an invasive behavior.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Control, Staining, Phospho-proteomics, Solvent, Comparison, Inhibition, Disruption

Journal: Cancer Science

Article Title: Importance of SUMOylation Consensus Sequence of MAFK in Regulating EMT , Tumor Growth, Stemness, and Drug Resistance

doi: 10.1111/cas.70282

Figure Lengend Snippet: The MAFK SUMOylation potentially correlates with drug resistance in breast cancer cells. (A, B) Doxorubicin cytotoxicity assays and IC 50 values were measured in NMuMG‐mock, MAFK (WT) (K4, K10), and MAFK (EA) (EA10, EA12) cells (A), as well as in MDA‐MB‐231 with MAFK knockdown (siK2, siK3) (B). ** p < 0.01, **** p < 0.0001; nonlinear regression with 95% asymmetric confidence intervals. (C) Quantitative PCR (qPCR) analysis of Abcg2 expression in stable NMuMG cell lines treated with or without 0.25 μM doxorubicin for 24 h. (D) qPCR analysis of MAFK and ABCG2 in MDA‐MB‐231 cells treated with or without 0.25 μM doxorubicin for the indicated time points. **** p < 0.0001; two‐way ANOVA followed by Dunnett's multiple comparisons test. (E) qPCR analysis of MAFK and ABCG2 in MDA‐MB‐231 cells with MAFK knockdown (siK2, siK3). **** p < 0.0001; one‐way ANOVA followed by Tukey's multiple comparisons test. All qPCR values were normalized to β‐actin mRNA. All data are representative of three independent replicates. (F) Transcriptome data analysis of MAFK and ABCG2 in triple‐negative breast cancer patients from a clinical database who received Anthracycline treatment. The p value was calculated using ROC analysis.

Article Snippet: 293T, NMuMG, and MDA‐MB‐231 cells were purchased from American Type Culture Collection (ATCC).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: PBRM1-Dependent PBAF Targeting is Required for EMT and Metastasis in Breast Cancer

doi: 10.1101/2025.10.19.683137

Figure Lengend Snippet: (A) Schematic of NMuMG cells treatment with TGFβ1 and micrographs of associated morphological changes (4X magnification). (B and C) Representative transwell invasion assay images (B) and bar plot (C) of absorbance quantification of NMuMG sgCt and sg Pbrm1 cells with and without TGFβ1 treatment. n=3 technical replicates. Data are represented as mean ± SD. (D) Immunoblots of Pbrm1, E-Cadherin, and Vimentin in lysates of NMuMG vector control and sh Pbrm1 cells with TGFβ1 treatment at the indicated concentrations and incubation times. (E) Relative cell counts of NMuMG control and sh Pbrm1 cells with and without TGFβ1 treatment, normalized to untreated cells. Representative graph, n=3 biological replicates. Data are represented as mean ± SD. (F) Relative cell counts of NMuMG sgCt and sg Pbrm1 cells with and without TGFβ1 treatment, normalized to untreated cells. Representative graph, n=3 biological replicates. Data are represented as mean ± SD. (G) Representative flow cytometry density dot plots of Edu and PI staining in fixed NMuMG sgCt and sg Pbrm1 cells with and without 72h TGFβ1 treatment. The gating strategy for cells in different cell cycle stages (G0/G1, S, or G2/M) is indicated with boxes. (H) Bar plot of the percentage of cells in different cell cycle stages in NMuMG sgCt and sg Pbrm1 cells with TGFβ1 treatment for the indicated incubation times. Gating was performed as in (G). n=3 biological replicates. Data are represented as mean ± SD. (I) Representative flow cytometry density dot plots of AnnexinV and PI staining in unfixed NMuMG sgCt and sg Pbrm1 cells with and without 5d TGFβ1 treatment. The gating strategy for the percentage of live (PI-)/dead (PI+) and apoptotic (Annexin V+)/non-apoptotic (Annexin V-) cells is indicated with quadrants. (J) Bar plot of percentage of PI-live cells (left) and Annexin V+ apoptotic cells (right) in NMuMG sgCt and sg Pbrm1 cells with TGFβ1 treatment for the indicated incubation times. Gating was performed as in (I). n=3 biological replicates. Data are represented as mean ± SD. (K) Immunoblots of Pbrm1 and Cleaved-PARP (C-PARP) from lysates of NMuMG sgCt and sg Pbrm1 cells with and without TGFβ1 treatment for the indicated time periods. Statistical comparison was done using multiple unpaired t-tests with Holm-Sidak correction. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001

Article Snippet: NMuMG cells were purchased from ATCC and grown in DMEM (Corning) supplemented with 10% fetal bovine serum (Corning), 10 μg/mL Insulin (Sigma), 1% L-glutamine (Corning), 1% antibiotics (100 units/mL penicillin and 100 g/mL streptomycin; Corning), and 1% Sodium Pyruvate (Corning) The EGFR transformed NME cell line with stable luciferase expression has been reported before and was cultured similar to NMuMG cells as described above.

Techniques: Transwell Invasion Assay, Western Blot, Plasmid Preparation, Control, Incubation, Flow Cytometry, Staining, Comparison

Journal: bioRxiv

Article Title: PBRM1-Dependent PBAF Targeting is Required for EMT and Metastasis in Breast Cancer

doi: 10.1101/2025.10.19.683137

Figure Lengend Snippet: (A) Schematic representation of the PBAF and cBAF complexes with shared subunits in grey, PBAF-specific subunits in red, and cBAF specific subunits in blue. The reader domains encoded by the different subunits and their respective functions are indicated. (B) Venn diagram of Phf10 and Smarca4 ChIP-seq peaks from NMuMG sgCt cells with Pbrm1 and Dpf2 ChIP-seq peaks from NMuMG cells (GSE249211). Total number of peaks identified in ChIP-seq for each protein is indicated in parentheses. (C) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10, Pbrm1, Smarca4, and Dpf2 at Phf10 and Dpf2 ChIP-seq sites in NMuMG cells as described in (B). (D) Genomic feature distribution of the ChIP-seq peaks identified for Pbrm1, Phf10, Smarca4, and Dpf2 in NMuMG cells as described in (B). (E) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10 H3K4me3, H3K4me1, and H3K27ac enrichment at Phf10 ChIP-Seq sites in sgCt cells as described in (B). (F) Heatmap representation of PBAF subunit abundance in NMuMG sg Pbrm1 cells with and without TGFβ1 treatment identified by Phf10 IP-MS. (G) (left)Venn diagram of Phf10 ChIP-seq peaks in NMuMG sgCt and sg Pbrm1 cells. Total number of Phf10 ChIP-seq peaks identified in each condition is indicated in parentheses. (right) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10 in sgCt and sg Pbrm1 cells at Phf10 ChIP-seq sites in sgCt cells as described in (B). Peak summits are aligned at the center. (H) Venn diagram of Phf10 ChIP-seq peaks in untreated and TGFβ1-treated NMuMG sgCt cells. Total number of Phf10 ChIP-seq peaks identified in each condition is indicated in parentheses. (I) Metagene plots and heatmaps of Phf10 ChIP-seq enrichment in untreated and TGFβ1-treated NMuMG cells. Enrichment was plotted at Phf10 sites in untreated sgCt cells (top) and Phf10 sites unique to TGFβ-treated sgCt cells. Peak summits are aligned at the center. (J) (top) Venn diagram of Phf10 ChIP-seq peaks in NMuMG sgCt cells and sites of differential accessibility in sg Pbrm1 cells compared to sgCt cells. (bottom) The genomic feature distribution for sites of differential accessibility in sg Pbrm1 cells compared to sgCt cells (K) (top) Venn diagram of Phf10 ChIP-seq peaks in NMuMG sgCt cells with 48h TGFβ1 treatment and sites of differential accessibility in sg Pbrm1 cells compared to sgCt cells with 48h TGFβ1 treatment. (bottom) The genomic feature distribution for sites of differential accessibility in sg Pbrm1 cells compared to sgCt cells with 48h TGFβ1 treatment. (L) Metagene plots and heatmaps of Phf10 ChIP-seq and ATAC-seq enrichment at sites of differential accessibility in sg Pbrm1 cells compared to sgCt cells under untreated (left) and 48h TGFβ1-treated (right) conditions. (M) Genomic tracks of Phf10 ChIP-seq and ATAC-seq enrichment at the Tnfsf13b locus in sgCt and sg Pbrm1 cells in untreated (top) and 48h TGFβ1-treated (bottom) conditions.

Article Snippet: NMuMG cells were purchased from ATCC and grown in DMEM (Corning) supplemented with 10% fetal bovine serum (Corning), 10 μg/mL Insulin (Sigma), 1% L-glutamine (Corning), 1% antibiotics (100 units/mL penicillin and 100 g/mL streptomycin; Corning), and 1% Sodium Pyruvate (Corning) The EGFR transformed NME cell line with stable luciferase expression has been reported before and was cultured similar to NMuMG cells as described above.

Techniques: ChIP-sequencing, Protein-Protein interactions

Journal: bioRxiv

Article Title: PBRM1-Dependent PBAF Targeting is Required for EMT and Metastasis in Breast Cancer

doi: 10.1101/2025.10.19.683137

Figure Lengend Snippet: (A) Volcano plot of TF consensus motifs enriched in regions of differential accessibility in NMuMG sg Pbrm1 relative to sgCt cells with 48h TGFβ1 treatment (FDR<0.001). (B) Metagene plots and heatmaps of ChIP-seq enrichment of Fosl2 (left), Fosb (center) and Atf3 (right) in sgCt and sg Pbrm1 cells in both untreated and 48h TGFβ1-treated conditions. Enrichment was plotted for corresponding TF binding sites compiled from all conditions. (C) Venn diagram of Phf10 ChIP-Seq sites with Fosl2 (left), Fosb (center) and Atf3 (right) ChIP-seq sites in sgCt cells with 48h TGFβ1 treatment. Total number of peaks identified in ChIP-seq for each protein is indicated in parentheses. To the right of each Venn diagram are the metagene plots and heatmaps of ChIP-seq enrichment of Phf10 with Fosl2 (left), Fosb (center) or Atf3 (right) at the Phf10 (top) and associated TF (bottom) binding sites. (D) Genomic feature distribution of the Fosl2, Fosb, and Atf3 ChIP-seq peaks. (E) Genomic tracks of Phf10 and Atf3 ChIP-seq enrichment in sgCt and sg Pbrm1 cells at Tnfsf13b locus in untreated (top) and 48h TGFβ1-treated (bottom) conditions. (F) Scatter plot of the change in expression for DEGs from sg Pbrm1 vs sgCt with 48h TGFβ1 plotted against DEGs from sh Atf3 vs shScr with 48h TGFβ1. Each data point in the scatter plot represents the log2FC expression value of a single gene in the indicated comparison, x axis : sh Atf3 vs shCt, y axis : sg Pbrm1 vs sgCt. The degree of correlation was calculated using all DEGs. (G) Venn diagram of overlap between DEGs in sg Pbrm1 vs sgCt and DEGs from sh Atf3 vs shScr, both with 48h TGFβ1 treatment. Total number of genes from each condition is indicated in parentheses. The top overrepresented GO terms for the set of genes increased by both sg Pbrm1 and sh Atf3 (top) or decreased by both sg Pbrm1 and sh Atf3 (bottom) were identified using Enrichr pathway analysis. (H) Absolute cell counts of control and sh Atf3 cells with and without 5 ng/mL TGFβ1 treatment. 0.9 million cells were seeded for each cell line and cell counts were taken on day 3 and 6. Representative graph, n=3 biological replicates. Data are represented as mean ± SD. (I) Heatmap representation of the differential accessibility of consensus TF motifs in NMuMG sg Pbrm1 cells with and without TGFβ1 treatment performed using diffTF analysis. Significant differences in weighted means between the groups are shown. (J) Intersection of Phf10 ChIP-seq peaks in NMuMG sgCt cells with Irf1 ChIP-seq peaks in 48h TGFβ1-treated NMuMG cells from a published Irf1 ChIP-Seq dataset (GSE141501). Total number of peaks identified in ChIP-seq for each protein is indicated in parentheses. (K) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10 from 48h TGFβ1-treated NMuMG sgCt cells and Irf1 from 48h TGFβ1-treated NMuMG cells from a published dataset at Phf10 and Irf1 shared sites. (L) Genomic tracks of ChIP-seq enrichment of Phf10, Atf3, H3K14ac, and Irf1 in untreated and 48h-TGFβ1 treated NMuMG sgCt at Il15 locus. (M) Intersection of Phf10 ChIP-seq peaks in NMuMG sgCt cells with Snai1 ChIP-seq peaks in pBI3G mouse mesenchymal breast cancer cells from a published dataset (GSE61198). Total number of peaks identified in ChIP-seq for each protein is indicated in parentheses. (N) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10 from 48h TGFβ1-treated NMuMG sgCt cells and Snai1 from pBI3G mouse mesenchymal breast cancer cells at Phf10 and Snai1 shared sites. (O) Genomic tracks of ChIP-seq enrichment at Snai2 locus of Phf10, Atf3, and H3K14ac in untreated and 48h-TGFβ1 treated NMuMG sgCt cells and Snai1 ChIP-Seq enrichment in pBI3G mouse mesenchymal breast cancer cells. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001

Article Snippet: NMuMG cells were purchased from ATCC and grown in DMEM (Corning) supplemented with 10% fetal bovine serum (Corning), 10 μg/mL Insulin (Sigma), 1% L-glutamine (Corning), 1% antibiotics (100 units/mL penicillin and 100 g/mL streptomycin; Corning), and 1% Sodium Pyruvate (Corning) The EGFR transformed NME cell line with stable luciferase expression has been reported before and was cultured similar to NMuMG cells as described above.

Techniques: ChIP-sequencing, Binding Assay, Expressing, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Natural Polysaccharide-Based Nanoparticles Enhance Intracellular Delivery and Cytotoxicity of Antrodia camphorata in Breast Cancer Cells

doi: 10.3390/ijms26178420

Figure Lengend Snippet: Cell viability analysis using WST assay for ( a ) NMuMG, ( b ) MDA-MB-231, and ( c ) MCF-7 cells treated with deionized (DI) water, unloaded (UL)-NPs, AC+DI water, and AC8-NPs at two concentrations (107.3 µg/mL and 241.7 µg/mL) for 24, 48, and 72 h. Control samples were untreated. Data represent the mean ± standard deviation (SD) ( n = 6) from three independent experiments. * p < 0.05 and ** p < 0.001 compared to the DI water group. ( d ) IC 50 values of AC8-NPs after 72 h incubation of MDA-MB-231 and MCF-7 cell lines. AC, Antrodia camphorate ; NPs, nanoparticles.

Article Snippet: NMuMG murine mammary gland cells, MDA-MB-231 human breast cancer cells, and MCF-7 human breast epithelial cells were obtained from the American Type Culture Collection.

Techniques: WST Assay, Control, Standard Deviation, Incubation

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: Normal murine mammary gland (NMuMG) epithelial cells were obtained from American Type Culture Collection (ATCC Cat# CRL‐1636, RRID: CVCL_0075) and were maintained in Dulbecco's Modified Eagle Medium (DMEM; Corning) with 10% (v/v) fetal bovine serum (FBS; Atlanta Biologicals), 50 μg/mL gentamicin (Gibco), and 10 μg/mL insulin (Sigma Aldrich).

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: Normal murine mammary gland (NMuMG) epithelial cells were obtained from American Type Culture Collection (ATCC Cat# CRL‐1636, RRID: CVCL_0075) and were maintained in Dulbecco's Modified Eagle Medium (DMEM; Corning) with 10% (v/v) fetal bovine serum (FBS; Atlanta Biologicals), 50 μg/mL gentamicin (Gibco), and 10 μg/mL insulin (Sigma Aldrich).

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: Normal murine mammary gland (NMuMG) epithelial cells were obtained from American Type Culture Collection (ATCC Cat# CRL‐1636, RRID: CVCL_0075) and were maintained in Dulbecco's Modified Eagle Medium (DMEM; Corning) with 10% (v/v) fetal bovine serum (FBS; Atlanta Biologicals), 50 μg/mL gentamicin (Gibco), and 10 μg/mL insulin (Sigma Aldrich).

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: Normal murine mammary gland (NMuMG) epithelial cells were obtained from American Type Culture Collection (ATCC Cat# CRL‐1636, RRID: CVCL_0075) and were maintained in Dulbecco's Modified Eagle Medium (DMEM; Corning) with 10% (v/v) fetal bovine serum (FBS; Atlanta Biologicals), 50 μg/mL gentamicin (Gibco), and 10 μg/mL insulin (Sigma Aldrich).

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: Normal murine mammary gland (NMuMG) epithelial cells were obtained from American Type Culture Collection (ATCC Cat# CRL‐1636, RRID: CVCL_0075) and were maintained in Dulbecco's Modified Eagle Medium (DMEM; Corning) with 10% (v/v) fetal bovine serum (FBS; Atlanta Biologicals), 50 μg/mL gentamicin (Gibco), and 10 μg/mL insulin (Sigma Aldrich).

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

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