Journal: Bone & Joint Research

Article Title: Piezo1 drives fibroblast activation in epidural fibrotic remodelling via the ET-1/HIF-1α pathway

doi: 10.1302/2046-3758.158.BJR-2025-0662.R1

Figure Lengend Snippet: The hypoxia-inducible factor 1-alpha (HIF-1α)/transforming growth factor beta 1 (TGF-β1)/Smad3 pathway functions downstream of Piezo1 in the activation of fibroblasts. a) Western blot analysis of NIH/3T3 fibroblasts treated with DMSO or Yoda1 (n = 3). b) Western blot analysis of NIH/3T3 fibroblasts treated with Yoda1 and the HIF-1α inhibitor BAY 87-2243 (n = 3). c) and d) Western blot analysis of NIH/3T3 fibroblasts cultured with type I rat tail collagen and the HIF-1α inhibitor BAY 87-2243 (n = 3). e) and f) Western blot analysis of NIH/3T3 fibroblasts cultured with type I rat tail collagen and the TGF-β1 inhibitor SB-431542 (n = 3). g) Western blot analysis of NIH/3T3 fibroblasts cultured with type I rat tail collagen and the Smad3 inhibitor SIS 3 (n = 3). h) and i) Western blot analysis of NIH/3T3 fibroblasts cultured with type I rat tail collagen and the Piezo1 inhibitor GsMTX4 (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, independent-samples t -test (two groups) or analysis of variance (multiple groups).

Article Snippet: Alternatively, the following drugs were used to stimulate NIH/3T3 cells: Yoda1 (25 μM; HY-P1410), a chemical activator of Piezo1; BAY 87-2243 (1 μM; HY-15836), an inhibitor of hypoxia-inducible factor 1-alpha (HIF-1α); SB-431542 (10 μM; HY-10431), an inhibitor of TGF-β1; SIS 3 (10 μM; HY-13013), an inhibitor of Smad3; GsMTX4 (2.5 μM; HY-P1410); Nimodipine (60 μM; HY-B0265), a calcium channel blocker; and Bosentan (1 μM; HY-A0013, all MedChemExpress), an endothelin-1 (ET-1) receptor antagonist.

Techniques: Activation Assay, Western Blot, Cell Culture

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: Morphological characterization of iEPDCs after five days of culture with SB431542, NoAdditive and TGFβ3. Phalloidin-F-actin staining illustrated distinct morphological phenotypes of iEPDCs following treatment. TGFβ3-treated iEPDCs exhibited an elongated spindle-shaped morphology, whereas SB431542-treated cells displayed a cobblestone-like appearance. Cells cultured without any additive showed an intermediate morphology with features of both TGFβ3- and SB431542-treated phenotypes. Nuclei were counterstained with DAPI.

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques: Staining, Cell Culture

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: TGFβ3 treatment reduces epithelial-like features in iEPDCs. iEPDCs were cultured for five days under SB431542, NoAdditive or TGFβ3 conditions. ( A ) Immunocytochemical staining of iEPDCs demonstrates lower expression of epithelial-associated markers MSLN and CRIP1 in iEPDCs treated with TGFβ3 for five days compared with SB431542-treated cells, indicating a loss of epithelial characteristics. iEPDC cultures under the NoAdditive condition show intermediate levels of these markers, resembling an expression pattern between SB431542- and TGFβ3-treated cells. Nuclei were counterstained with DAPI. ( B ) Quantification of nuclear WT1 relative fluorescence intensity confirms significantly lower levels of WT1 in TGFβ3-treated iEPDCs compared to SB431542-treated cells. Relative fluorescence intensities of MSLN and CRIP1, determined by threshold-based analysis, were similarly decreased following TGFβ3 treatment compared to SB431542-treated iEPDCs. All data are shown as mean ± SEM, RM one-way ANOVA with Tukey’s post hoc test; p * < 0.05; p ** < 0.01; n = 3 donors.

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques: Cell Culture, Staining, Expressing, Fluorescence

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: TGFβ3 enhances mesenchymal-like features in iEPDCs. iEPDCs were cultured for five days under SB431542, NoAdditive or TGFβ3 conditions. ( A ) Immunocytochemical staining of iEPDCs shows that VIM and CD105/Endoglin expression levels are elevated in iEPDCs treated with TGFβ3 compared with SB431542-treated cells. iEPDCs cultured with NoAdditive show intermediate levels of both markers. The increased abundance and filamentous organization of VIM in TGFβ3-treated iEPDCs reflect cytoskeletal remodeling associated with a mesenchymal-like phenotype. Nuclei were counterstained with DAPI. ( B ) Quantification of VIM and CD105 relative mean fluorescence intensity confirms higher levels in TGFβ3-treated iEPDCs compared to SB431542-treated cells. All data are shown as mean ± SEM, RM one-way ANOVA with Tukey’s post hoc test; p * < 0.05; p ** < 0.01; n = 3 donors.

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques: Cell Culture, Staining, Expressing, Fluorescence

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: Principal component analysis (PCA) of iEPDCs and related cell populations. PCA of iEPDCs cultured for five days under SB431542-, NoAdditive- or TGFβ3-conditions, together with hCSCs and hPSC-derived epicardial cells, reveals that all cell populations cluster independently.

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques: Cell Culture, Derivative Assay

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: Differential gene expression in TGFβ3- versus SB431542-treated iEPDCs after five days of treatment. Volcano plot of differentially expressed genes shows upregulation of 287 genes in TGFβ3-treated iEPDCs and upregulation of 293 genes in SB431542-treated iEPDCs. The boxes highlight genes associated with EPDCs and the epicardium. The complete list of DEGs is provided in .

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques: Gene Expression

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: ( A ) Volcano plot of differentially expressed genes in EPDCs treated with SB431542 vs. NoAdditive. ( B ) Volcano plot of differentially expressed genes in EPDCs treated with TGFβ3 vs. NoAdditive. The boxes highlight genes associated with EPDCs and the epicardium. Volcano plot showing DEGs after 5 days of treatment. Complete differential expression results for the respective comparisons are provided in .

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques: Quantitative Proteomics

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: Heatmap of the top 200 differentially expressed genes in iEPDCs treated with SB431542, NoAdditive and TGFβ3 for five days. The dashed box indicates genes that are mainly upregulated in TGFβ3-treated iEPDCs and downregulated under SB431542 conditions, whereas the solid box highlights genes that are mainly upregulated in SB431542-treated iEPDCs and mainly downregulated in TGFβ3-treated cells. A version of the heatmap displaying all gene labels is provided in .

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques:

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: KEGG and GO enrichment analyses of TGFβ3-treated vs. SB431542-treated iEPDCs after five days of treatment. ( A ) Upregulated KEGG terms in TGFβ3-treated iEPDCs compared with SB431542-treated cells. ( B ) Upregulated KEGG terms in SB431542-treated iEPDCs compared with TGFβ3-treated cells. Upregulated GO terms in TGFβ3-treated iEPDCs compared with SB431542-treated in ( C ) cellular component, ( D ) molecular function and ( E ) biological process. ( F ) Upregulated biological process GO terms in SB431542-treated iEPDCs compared with TGFβ3-treated cells. For each analysis, the top 15 significantly enriched pathways/terms are displayed.

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques:

Journal: Cells

Article Title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

doi: 10.3390/cells15141313

Figure Lengend Snippet: TGFβ3-treated mesenchymal-like iEPDCs show a higher relative closure ratio than SB431542-treated cells after 8 h. iEPDCs were cultured for five days under SB431542, NoAdditive or TGFβ3 conditions before scratching. ( A ) Representative images of a scratch after 0 h and 8 h. ( B ) Relative closure ratio indicates a higher migration rate of TGFβ3-treated iEPDCs compared to SB431542-treated cells. Data are shown as mean ± SEM, RM one-way ANOVA with Tukey’s post hoc test; p * < 0.05; p ** < 0.01; n = 5 donors.

Article Snippet: The cells were then plated on dishes coated with 0.1% gelatine A (Sigma-Aldrich, Taufkirchen, Germany) and cultured with EPDC-medium + SB431542, a TGFβRI inhibitor, containing DMEM low-glucose (10567014; Thermo Fisher Scientific, Schwerte, Germany) and M199 (31150022; Thermo Fisher Scientific, Schwerte, Germany) mixed at a ratio of 1:1, supplemented with 10% fetal calf serum (Sigma-Aldrich, Taufkirchen, Germany), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany) and 10 μM SB431542 (MedChemExpress, Sollentuna, Sweden) to maintain the epithelial-like phenotype.

Techniques: Cell Culture, Migration