Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Structural and biological characterization of SIS and UBM-SIS meshes and isolated MBVs. A) Surface and cross-sectional morphology of SIS and UBM-SIS meshes by SEM. B) Quantification of mesh thickness (n = 10). C) Pore size and porosity analysis of mesh (n = 10). D) Live/dead staining of fibroblasts on SIS and UBM-SIS at day 1, 4, and 7. (E) CCK-8 assay of fibroblast viability on meshes (n = 5). F) Immunofluorescence staining of fibroblasts (TGF-β1, day 3), SMCs (phalloidin, day 7) and HUVECs (CD31, day 14) on SIS and UBM-SIS meshes, and SEM and DAPI staining of SMCs (day 21) coverage and cellular infiltration. White dashed lines delineate the upper and lower boundaries of the ECM scaffold. The yellow dashed line indicates the infiltration depth, defined as the distance from the scaffold surface to the DAPI-positive nucleus formed as a cellular floor and used for quantitative analysis. G) Quantification of TGF-β1, cytoskeletal area (phalloidin) and CD31 expression (n = 5). H) Quantification of cellular infiltration across mesh thickness (n = 5). I) Schematic of ECM components retained in decellularized ECM mesh. J) H&E and Masson's trichrome staining of mesh. K) Residual DNA quantification. L) Analysis of cytokine and growth factor profiling upon mesh-specific difference (n = 4). M) Workflow for MBV isolation and analysis. N) SEM images of MBV embedded on ECM. O) TEM images of MBV morphology. P) NTA analysis of MBV (n = 4). Q) Western blot detection of exosomal markers in MBV. Data are presented as mean ± SD. Each dot represents an independent sample. Statistical significance was determined using two-tailed unpaired Student's t-test for comparisons between two groups (B, C, and K), or one-way ANOVA followed by Tukey's post hoc test for multiple comparisons (E, G, and H), where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Isolation, Pore Size, Staining, CCK-8 Assay, Immunofluorescence, Expressing, Western Blot, Two Tailed Test

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Bioactivity and immunomodulatory properties of MBVs derived from SIS and UBM-SIS meshes. A) Schematic illustration of MBV-regulated cellular activities during ECM remodeling via their interactions with fibroblasts, SMCs, HUVECs, and macrophages to validate MBVs as bioactive components embedded within parent ECM. Nuclei are labeled with DAPI (blue); PKH26 (red) marks MBVs; phalloidin (green) stains F-actin. B) Immunofluorescence staining of fibroblasts (TGF-β1, collagen I), SMCs (phalloidin), and HUVECs (CD31) after treatment with SIS MBVs or UBM-SIS MBVs. C) Quantification of fluorescence signal area per cell for respective markers (n = 5). D) Western blot analysis of marker proteins in MBV-treated cells. E) Relative protein expression levels normalized to GAPDH (n = 3). F) Schematic of macrophage polarization model with/without MBV treatment. G) Immunostaining of macrophages for F4/80, iNOS (M1-like), and Arg-1 (M2-like) under different stimulations and MBV-treated conditions. H) Quantification of mean fluorescence intensity (MFI) of iNOS and Arg-1 (n = 5). I) Western blot analysis of pro- and anti-inflammatory markers in MBV-treated macrophages and LPS + IFN-γ-treated macrophages (control). J) Quantification of relative protein levels (n = 3). K) Heatmap of RT-qPCR analysis showing cytokine and ECM regulator gene expression in MBV-treated macrophages (n = 3). L) Representative fluorescence images of DCFH staining in macrophages following different treatments. M) Quantification of DCFH fluorescence area per cell (n = 5). Data are presented as mean ± SD. Each dot represents an independent biological replicate. Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Derivative Assay, Labeling, Immunofluorescence, Staining, Fluorescence, Western Blot, Marker, Expressing, Immunostaining, Control, Quantitative RT-PCR, Gene Expression

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Functional comparison of MBVs derived from SIS and UBM in modulating inflammation, angiogenesis, and matrix remodeling. A) Schematic of macrophage polarization model under LPS + IFN-γ stimulation with or without MBV treatment. B) Immunofluorescence staining of iNOS (red) and Arg-1 (green) in macrophages treated with SIS MBVs, UBM MBVs, or UBM-SIS MBVs. C) Quantification of mean fluorescence intensity (MFI) of iNOS and Arg-1 (n = 5). D–G) RT-qPCR analysis of pro- (( TNF-α, IL-6 ) and anti-inflammatory ( IL-10, TGF-β1 ) cytokine gene expression in MBV-treated macrophages (n = 4). H) Schematic of analysis of MBV-treated HUVECs and fibroblasts cultured in Matrigel. I) 3D immunostaining of CD31 + tube-like structures in HUVECs after MBV treatment. J–K) Quantification of tube-like area percentage and number of tube-like structures per field (n = 6). L) 3D two-photo images of TGF-β1 expression in fibroblasts cultured with different MBVs. M) Quantification of TGF-β1-positive volume percentage in fibroblasts (n = 4). N-P) Western blot analysis of NF-κB and STAT3 pathway proteins in MBV-treated macrophages, angiogenic signaling proteins (AKT, ERK1/2) in MBV-treated HUVECs, TGF-β/Smad signaling pathway in MBV-treated fibroblasts. Quantification of respective signaling molecules (n = 3). Data are presented as mean ± SD. Each dot represents an independent biological replicate. Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Functional Assay, Comparison, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR, Gene Expression, Cell Culture, Immunostaining, Expressing, Western Blot

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Inflammatory immune responses following mesh implantation. A) Schematic illustration and representative macroscopic images of seroma tissues collected from the explants at 1 week. B) ELISA analysis of cytokines in the seroma fluid after 1 week (n = 4). C) Representative immunofluorescence images of CD11b + cell infiltration in mesh at 1 and 4 weeks. Scale bars: left, 1000 μm; right, 50 μm. D–E) Quantification of CD11b + cell density (n = 5, 4 samples per rat). F) Representative immunofluorescence images of CD68 (yellow), CD86 (green), and CD206 (red) staining of SIS and UBM-SIS at 1 and 4 weeks. Scale bars: top, 1000 μm; bottom, 50 μm. G–H) Quantification of CD68 + macrophage infiltration and M2-like/M1-like phenotypic distribution at 1 week (n = 5). I) Statistical comparison of M2-like/M1-like ratios between SIS and UBM-SIS groups (n = 5). J–K) CD68 + macrophage infiltration and CD206 + /CD86 + phenotypic distribution at 4 weeks (n = 5, 4 samples per rat). L) Quantification of M2-like/M1-like ratios at 4 weeks (n = 5, 4 samples per rat). M−O) Representative immunofluorescence images of iNOS and Arg-1 at tissue-mesh interfaces at 1 and 4 weeks, with quantitative analysis of positive area (n = 5, with 4 samples per rat). Scale bars: 50 μm. P) RT-qPCR analysis of pro- (( TNF-α, IL-6 ) and anti-inflammatory ( IL-10, TGF-β1 ) cytokine gene expression in SIS and UBM-SIS explants. Q) Schematic summary of immune response transition induced by SIS versus UBM-SIS MBV-containing meshes over 4 weeks. The asterisk indicates the implanted mesh. Data are presented as mean ± SD. For (B, I, L, and P), mean value of each rat (n = 5) was used for statistical comparisons. For (D, E, G, H, J, K, N, and O), each dot represents one section-level sample, where the value of each animal for statistical comparisons was obtained by averaging measurements from 4 samples. Statistical comparisons were performed within each time point using two-tailed unpaired Student's t-test, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Comparison, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: ECM remodeling and mechanical evaluation of meshes and explants. A) Representative immunofluorescence images showing collagen I (red) and collagen III (green) deposition in the center and interface regions of SIS and UBM–SIS explants at 8 weeks. Scale bars: overview = 1000 μm, magnified images = 100 μm. B–E) Quantification of total collagen (I + III), collagen I, and collagen III positive expression and collagen I/III ratio in the center and interface regions (n = 5, 4 samples per rat). F–G) Polar plot of collagen fiber orientation in the center and interface regions analyzed by orientation distribution. H) Orientation coherency of collagen fibers in the center and interface regions (n = 5, 4 samples per rat). I) Aspect ratio analysis indicating collagen fibril anisotropy (n = 4). J–K) Representative immunofluorescence staining of TGF-β1 and α-SMA at 8 weeks and corresponding quantification of positive area (n = 5, 4 samples per rat). L, M) Mechanical characterization of meshes and explants showing ultimate tensile strength and elongation at break across different time points (n = 4). N) Comparison of tensile strength and elongation of explants with native abdominal wall components, including posterior and anterior rectus sheath, linea alba, peritoneum, and transversalis fascia. The asterisk indicates the implanted mesh. Data are presented as mean ± SD. For (B-E, H, and K), mean value of each rat (n = 5) was used for statistical comparisons. Each dot represents one section-level sample, where the value for each animal was obtained by averaging measurements from 4 samples. For (I, L, and M), mean value of each rat (n = 4) was used for statistical comparisons. Statistical significance was determined using two-tailed unpaired Student's t-test, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunofluorescence, Expressing, Staining, Comparison, Two Tailed Test

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Transcriptomic analysis of explants from different groups after 1 week and comparative analysis of MBV and ECM properties. A) Schematic illustration of tissues collected from the explants at 1 week for distinct signaling pathway analysis. B) Volcano plot showing differentially expressed genes (DEGs) between UBM-SIS and SIS groups (n = 3 per group). C) Heatmap of DEGs between SIS and UBM-SIS groups (red: upregulated, blue: downregulated; cutoff >1.0; n = 3). D) KEGG pathway enrichment analysis of downregulated genes in UBM-SIS compared to SIS. E) Reactome pathway enrichment analysis of downregulated genes in UBM-SIS compared to SIS. F–H) GSEA demonstrating altered gene signatures related to NET formation, NF-κB pathway and cytokine-cytokine receptor interaction. I) Radar plot comparing SIS- and UBM-derived MBVs. The five axes represent key pathways involved in angiogenesis ( ERK ), vascularization ( AKT ), inflammation ( p65 ), immunomodulation ( STAT3 ), and remodeling ( TGF-β/Smad ). J) Radar plot summarizing ECM in vivo performance at 1 and 4 weeks. The five axes represent essential features in ECM remodeling, including adhesion, angiogenesis, inflammation, immunomodulation, and collagen deposition.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Derivative Assay, In Vivo

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: EHF promotes lung cancer cell malignancy and M2 macrophage polarization. A549 and H520 cells were transfected with control (Ctrl) or EHF-knockdown (KD-EHF) plasmids. (A-B) Western blot and quantification of EHF protein levels in A549 and H520 cells. (C-D) EdU staining and quantification of EdU + cells in A549 and H520 cells, assessing proliferation. (E-H) Glucose uptake and lactate production assays were conducted using commercial kits to evaluate glycolytic output. (I-J) Flow cytometric analysis of apoptosis. (K-L) Transwell assay was employed to examine cell migration. (M − P) THP-1 cells were differentiated into THP-1-M0 macrophages with 100 ng/mL PMA for 24 h. Culture supernatants from Ctrl- or KD-EHF–transfected A549 and H520 cells were collected and co-incubated with THP-1-M0 macrophages. (M − N) Flow cytometric analysis was performed to assess the proportion of CD206 + M2-polarized macrophages. (O–P) ELISA was conducted to quantify the secretion of M2-associated cytokines (TGF-β1, IL-10, and VEGFA) in the supernatants of THP-1-M0 macrophages. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Transfection, Control, Knockdown, Western Blot, Staining, Transwell Assay, Migration, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: Exosomal EHF reprograms THP-1-M0 macrophages to promote lung cancer cell malignancy and angiogenesis via paracrine signaling. (A) Schematic of the paracrine co-culture system: Exosomes from control (Ctrl) or EHF-knockdown (KD-EHF) A549/H520 cells were co-incubated with THP-1-M0 macrophages. The medium from these macrophages was used to treat A549/H520 cells or HUVECs. (B-E) THP-1-M0 macrophages were treated with three conditions: blank control (no exosome), exosomes from control-transfected A549/H520 cells (Exo Ctrl ), or exosomes from EHF-knockdown A549/H520 cells (Exo KD−EHF ). (B–C) Flow cytometry analysis of CD206 + M2 macrophage proportions in THP-1-M0 cells. (D-E) ELISA measurement of M2-associated cytokines (TGF-β1, IL-10, and VEGFA). (F-M) The medium from exosome treated macrophages was then used to incubate A549/H520 cells or HUVECs. (F-G) EdU staining and quantification of EdU + proliferating A549 and H520 cells. (H–I) Flow cytometric analysis of apoptosis in A549 and H520 cells. (J-K) Transwell migration assays and quantification of migrated A549 and H520 cells. (L-M) Tube formation assays and quantification of tube numbers in HUVECs. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Co-Culture Assay, Control, Knockdown, Incubation, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Migration

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: RNF41 mediates exosomal EHF-driven pro-tumorigenic effects in lung cancer. (A) Schematic of the rescue co-culture system: THP-1-M0 macrophages (control or RNF41-overexpressing) were incubated with exosomes from control (Exo Ctrl ) or EHF-knockdown (Exo KD−EHF ) A549/H520 cells. The medium from these co-cultures was used to treat A549/H520 cells or HUVECs. (B–C) Flow cytometry was used to analyze the proportion of CD206 + M2-polarized macrophages in THP-1-M0 cells. (D-E) ELISA was employed to quantify the levels of M2-associated cytokines (TGF-β1, IL-10, and VEGFA) in the CM from macrophages. (F-G) EdU assays were carried out to measure the proliferation of A549 and H520 cells. (H–I) Annexin V/PI flow cytometry was used to assess the apoptosis of A549 and H520 cells treated with CM. (J-K) Transwell assays were performed to evaluate the migration of A549 and H520 cells. (L-M) Tube formation assays were conducted on HUVECs treated with CM. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Co-Culture Assay, Control, Incubation, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Migration

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: EHF knockdown inhibits tumor growth and downregulates M2-associated molecules in v ivo. Nude mice were subcutaneously implanted with Ctrl or KD-EHF A549 cells. (A) Tumor volume was measured every 5 days over 30 days. (B) Tumor weight. (C) Representative images of excised tumors. (D) Western blot analysis of RNF41, CD206, TGF-β1, and VEGFA expression levels in tumor tissues. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Knockdown, Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Intestinal epithelial PIEZO1 regulates macrophage-to-myofibroblast transition via epithelial-derived TGF-α signaling in Crohn’s disease

doi: 10.1186/s12964-026-02941-w

Figure Lengend Snippet: Intestinal epithelial PIEZO1 regulates MMT via TGF-α signaling. A The production of TGF-β1 in the CM derived from Caco-2-shCTL or Caco-2-shPIEZO1 for 24h was assessed by ELISA (n = 15). P values were calculated using Student’s t-test. B Volcano plot of differentially expressed genes. C The production of TGF-α in the CM was assessed by ELISA (n = 21). P values were calculated using Student’s t-test. D Pharmacological modulation of PIEZO1 regulates TGF-α secretion. Caco-2-shCTL cells were cultured to 50% or 100% confluence and treated with the PIEZO1 agonist Yoda1 or inhibitor GsMTx4, as indicated. TGF-α levels in supernatant were measured by ELISA (n = 10). P values were calculated using one-way ANOVA and Tukey’s multiple comparisons test. E Rescue experiment demonstrating that TGF-α mediates PIEZO1-dependent macrophage activation. THP-1 macrophages were treated with CM derived from Caco-2-shCTL or Caco-2-shPIEZO1 cells at 50% or 100% confluence, as indicated. Recombinant TGF-α was added to KD-100-treated macrophages to restore signaling activity. Western blotting was performed to detect the COL1A2, α-SMA, p-NF-κB P65 and p-PKA (n = 5). P values were calculated using one-way ANOVA and Tukey’s multiple comparisons test. F THP-1 macrophages were treated with CM derived from Caco-2-shCTL cells at 50% or 100% confluence (denoted as 50 and 100). For neutralization experiments, CM collected at 100% confluence was pre-incubated with an anti–TGF-α neutralizing antibody or isotype IgG control prior to macrophage stimulation. Protein expression of ECM-associated markers and downstream signaling molecules was analyzed by Western blot (n = 5). P values were calculated using one-way ANOVA and Tukey’s multiple comparisons test. G Western blotting was performed to detect the COL1A2, α-SMA, p-NF-κB P65 and p-PKA in RAW264.7 with TGF-α (n = 5). P values were calculated using one-way ANOVA and Dunnett’s multiple comparison test. H Western blotting was performed to detect COL1A2, α-SMA, p-NF-κB P65 and p-PKA in BMDMs with TGF-α (10 ng/mL) (n = 4). This concentration was selected based on dose–response experiments. P values were calculated using Student’s t-test. I Immunofluorescence showing the expression of TGF-α in colonic mucosa between healthy controls and CD patients. (n = 4). P values were calculated using Student’s t-test. Data are represented as mean ± SD, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Scale bar = 50μm. CM conditions are defined as in Fig.

Article Snippet: THP-1 macrophages were treated with recombinant human TGF-α (MedChemExpress, USA; Cat. # HY-P7296), BMDMs and RAW264.7 cells were treated with recombinant mouse TGF-α (MedChemExpress, USA; Cat. # HY- P77857 ).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Recombinant, Activity Assay, Western Blot, Neutralization, Incubation, Control, Expressing, Comparison, Concentration Assay, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Intestinal epithelial PIEZO1 regulates macrophage-to-myofibroblast transition via epithelial-derived TGF-α signaling in Crohn’s disease

doi: 10.1186/s12964-026-02941-w

Figure Lengend Snippet: Intestinal epithelial PIEZO1 deficiency attenuates colonic fibrosis in a chronic DSS-induced colitis–associated fibrosis model. A Schematic diagram of the chronic DSS-induced colitis-associated fibrosis model. B Body weight changes during DSS treatment (n = 6 mice per group). Statistical significance was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. C Disease activity index (DAI) scores in control and CKO mice during the experimental period (n = 6 mice per group). Statistical significance was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. Data are presented as mean ± SD. D Representative colon length changes in the indicated group (n = 5). P values were calculated using one-way ANOVA and Tukey’s multiple comparisons test. E Serum TGF-α levels measured by ELISA (n = 4). P values were calculated using Student’s t-test. F Immunofluorescence showed the expression of TGF-α in colonic tissues of colonic fibrosis in a mouse model (n = 6). P values were calculated using Student’s t-test. G Representative images of Masson’s trichrome-stained sections in the indicated group and the quantified area of ECM deposition per high-powered field (n = 4). P values were calculated using Student’s t-test. H Immunofluorescence showing the expression of CD68 + COL1A2 + cells and CD68 + α-SMA + cells in colonic tissues in the indicated group (n = 4). P values were calculated using Student’s t-test. I Immunofluorescence showing the expression of CD68 + p-PKA + cells and CD68 + p-NF-κB P65 + cells in colonic tissues in the indicated group (n = 4). P values were calculated using Student’s t-test. DAPI (blue), CD68 (green), and target proteins (red). Data are represented as mean ± SD, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Scale bar = 50 μm

Article Snippet: THP-1 macrophages were treated with recombinant human TGF-α (MedChemExpress, USA; Cat. # HY-P7296), BMDMs and RAW264.7 cells were treated with recombinant mouse TGF-α (MedChemExpress, USA; Cat. # HY- P77857 ).

Techniques: Activity Assay, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing, Staining