tgf β  (Elabscience Biotechnology)

Journal: Bioactive Materials

Article Title: Mesenchymal stromal cells-loaded 3D radially aligned composite scaffold with potentiated paracrine signaling for sequential bone regeneration

doi: 10.1016/j.bioactmat.2026.02.059

Figure Lengend Snippet: Temporal analysis of the BMSC paracrine profile on different scaffolds. (A) Confocal microscopy images from Live/Dead fluorescence staining of BMSCs encapsulated within the PCL/HAp-GelMA/BMSCs scaffold after 1, 3, 5, and 14 d of 3D culture (live cells, green; dead cells, red). (B) The concentrations of key paracrine factors (TGF-β, PGE2, VEGF, HGF, and BMP-2) from BMSCs cultured in different scaffolds, quantified from culture supernatants at day 3 and day 7. (C) Corresponding relative mRNA expression levels of TGFB1, PTGS2, VEGFA, HGF, and BMP-2 in BMSCs at day 3 and day 7, as determined by qPCR analysis. Data are presented as mean ± SD (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns: not significant.

Article Snippet: ELISA kits for PGE2 (Cat. No. E-EL-0034), TGF-β (Cat. No. E-EL-0162), VEGF (Cat. No. E-EL-R2603), and HGF (Cat. No. E-EL-R0496) were purchased from Elabscience (Wuhan, China).

Techniques: Confocal Microscopy, Fluorescence, Staining, Cell Culture, Expressing

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: Molecular Therapy Oncology

Article Title: The miR-16-1-3p suppresses proliferation and invasiveness via the MDM2-p53 axis in TGF-β1 signaling in osteosarcoma

doi: 10.1016/j.omton.2026.201249

Figure Lengend Snippet: MDM2 augments the TGF-β1-induced effects on U2OS cells (A) Colony formation assay of U2OS cells treated with 5 ng/mL TGF-β1, with or without MDM2 overexpression. Images of crystal violet-stained cell colonies (left) and the number of colonies quantified (right) at 10 days after seeding. (B) SRB assay of cell proliferation: total cell mass (absorbance at 570 nm) was measured at 0, 24, 48, and 72 h after seeding 5,000 cells/well in 96-well plates. (C) Wound healing assay: images of wound area (left) taken at 0 and 24 h after the “scratch” was made, and its quantification (right). Scale bar, 300 μm. (D) Transwell assay: images at 20× magnification display crystal violet-stained cells on the outer membrane of the inner chamber (left). It also depicts the cell migration rate 24 h after seeding 100,000 cells/well in the upper Boyden chamber of a 24-well Transwell insert (right). Scale bars, 50 μm. Data are presented as mean ± SD. p values in (A, C, and D) were determined using one-way ANOVA. ∗∗∗ p < 0.001.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (Novoprotein, China) was diluted in complete medium to a final concentration of 5 ng/mL , and added directly to the culture medium as part of the experimental treatment.

Techniques: Colony Assay, Over Expression, Staining, Sulforhodamine B Assay, Wound Healing Assay, Transwell Assay, Membrane, Migration

Journal: Molecular Therapy Oncology

Article Title: The miR-16-1-3p suppresses proliferation and invasiveness via the MDM2-p53 axis in TGF-β1 signaling in osteosarcoma

doi: 10.1016/j.omton.2026.201249

Figure Lengend Snippet: Influences of miR-16-1-3p mimics on the TGF-β1-mediated effects in U2OS cells (A) Dual-luciferase reporter assay in HeLa cells co-transfected with miR-16-1-3p mimics (50 nM) and a luciferase construct containing the wild-type MDM2 3′ UTR, confirming direct targeting. (B) Western blot showing MDM2 protein levels in U2OS cells 24 h after transfection with miR-16-1-3p mimics (50 nM) or Scramble control. (C) Colony formation assay in U2OS cells co-transfected with miR-16-1-3p mimics (50 nM) or Scramble control and MDM2 overexpression plasmid or empty vector. After 24 h, 500 cells/well were seeded in 6-well plates and treated with TGF-β1 (5 ng/mL) during colony formation. (D) SRB assay of cell proliferation: total cell mass (absorbance at 570 nm) was measured at 0, 24, 48, and 72 h after seeding 5,000 cells/well in 96-well plates. (E) Wound healing assay: images of the wound area (left) taken at 0 and 24 h after the “scratch” was made, and its quantification (right). Scale bar, 300 μm. (F) Transwell assay: images at 20× magnification display crystal violet-stained cells on the outer membrane of the inner chamber (left). The cell migration rate 24 h after seeding 1,000 cells/cm 2 in the upper Boyden chamber (right). Scale bar = 50 μm. Data are presented as mean ± SD. p values in (A and B) were determined using the Student’s t test. p values in (C, E, and F) were determined using one-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (Novoprotein, China) was diluted in complete medium to a final concentration of 5 ng/mL , and added directly to the culture medium as part of the experimental treatment.

Techniques: Luciferase, Reporter Assay, Transfection, Construct, Western Blot, Control, Colony Assay, Over Expression, Plasmid Preparation, Sulforhodamine B Assay, Wound Healing Assay, Transwell Assay, Staining, Membrane, Migration

Journal: Molecular Therapy Oncology

Article Title: The miR-16-1-3p suppresses proliferation and invasiveness via the MDM2-p53 axis in TGF-β1 signaling in osteosarcoma

doi: 10.1016/j.omton.2026.201249

Figure Lengend Snippet: Impact of miR-16-1-3p on TGF-β1 downstream signaling (A) Western blot analysis of pSmad2/3, total Smad2/3, p21, E-cadherin, N-cadherin, and vimentin expression in U2OS cells treated with 5 ng/mL TGF-β1 for 24 h (left). Quantification of protein expression (right). GAPDH expression was used for a protein loading control. U2OS exposed to 5 ng/mL TGF-β1 and 50 nM of miR-16-1-3p mimics after 24 h with or without MDM2 overexpression. (B) Flow cytometry analysis of cell-cycle distribution. Quantification of TGF-β1-promoted S-phase (left), and G1 arrest in U2OS cells (right). (C) TGF-β1 promoted cytoskeletal reorganization and EMT-like phenotype indicated by phalloidin fluorescence staining of F-actin remodeling. Scale bars, 150 μm. (D) Western blot analysis of stemness-associated markers OCT4 and SOX2 under the indicated treatment conditions (left), with densitometric quantification normalized to GAPDH (right). (E) 3D tumorsphere assay embedded in collagen matrix. Spheroids were first formed in agarose for 48 h and then embedded in collagen for another 24 h to evaluate invasion. Invasion was evaluated after 24 h of serum deprivation. Scale bars, 200 μm. Quantification of migration rates (upper right) and proliferation rates (bottom right). (F) Western blot of E-cadherin, N-cadherin, vimentin, and p21 expressions (left). Quantification of protein expression (right). GAPDH expression was used for a protein loading control. Data are presented as mean ± SD. p values in (A) were determined using two-way ANOVA. p values in (B, D, E, and F) were determined using one-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (Novoprotein, China) was diluted in complete medium to a final concentration of 5 ng/mL , and added directly to the culture medium as part of the experimental treatment.

Techniques: Western Blot, Expressing, Control, Over Expression, Flow Cytometry, Fluorescence, Staining, Migration

Journal: Molecular Therapy Oncology

Article Title: The miR-16-1-3p suppresses proliferation and invasiveness via the MDM2-p53 axis in TGF-β1 signaling in osteosarcoma

doi: 10.1016/j.omton.2026.201249

Figure Lengend Snippet: Impact of miR-16-1-3p on the functional output of TGF-β1 signaling (A) Western blot analysis and bubble plot quantification of U2OS cells transfected with 50 nM miR-16-1-3p mimic, 1 μg MDM2 overexpression plasmid, or control vector. After 24 h, cells were treated with 5 ng/mL TGF-β1 for an additional 24 h. (B) qPCR analysis of p21 mRNA expression in U2OS cells transfected for 24 h with miR-16-1-3p mimic (50 nM) or MDM2 overexpression plasmid, with or without TGF-β1 stimulation (5 ng/mL, 24 h). (C) Dual immunofluorescence staining of pSmad2/3 and p53 (left) and the number of pSmad2/3-positive vs. pSmad2/3-p53 double-positive cells (right) in response to TGF-β1 stimulation. Scale bars, 150 μm. (D) Bright-field imaging of pSmad2/3-positive but p53-negative cells (left) and cells co-expressing pSmad2/3 and p53 (right), supporting the notion of functional divergence based on signal combination. Scale bar, 200 μm. (E) Schematic model: in the presence of functional p53, TGF-β1-induced pSmad2/3 cooperates with p53 to activate p21 expression, leading to growth suppression. However, when p53 is repressed by MDM2, TGF-β1 signaling favors EMT and vimentin upregulation, promoting migration. miR-16-1-3p counters this effect by targeting MDM2, restoring p53 activity, and redirecting TGF-β1 output toward tumor suppression. Data are presented as mean ± SD. p values in (A and B) were determined using one-way ANOVA. p values in (C) were determined using two-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (Novoprotein, China) was diluted in complete medium to a final concentration of 5 ng/mL , and added directly to the culture medium as part of the experimental treatment.

Techniques: Functional Assay, Western Blot, Transfection, Over Expression, Plasmid Preparation, Control, Expressing, Immunofluorescence, Staining, Imaging, Migration, Activity Assay

Journal: Molecular Therapy Oncology

Article Title: The miR-16-1-3p suppresses proliferation and invasiveness via the MDM2-p53 axis in TGF-β1 signaling in osteosarcoma

doi: 10.1016/j.omton.2026.201249

Figure Lengend Snippet: miR-16-1-3p counteracts TGF-β1-mediated effects on metastasis and chemosensitivity of OS cells (A) Schematic representation of the chick embryo chorioallantoic membrane (CAM) assay. Stably Katushka2S-expressing U2OS cells were implanted onto the CAM to evaluate tumor growth and metastasis. Eggs were imaged at the indicated time points, and tumors were quantified at the experimental endpoint using a fluorescence imaging system. (B) Representative fluorescent and bright-field macroscopic images of tumor nodules formed on the CAM (left), with quantification of tumor volume shown on the right. (C) Representative fluorescent images of metastatic dissemination formed by Katushka2S-labeled U2OS cells in chick embryos under the indicated treatment conditions at the experimental endpoint. (D) Immunofluorescence analysis of Ki67 expression in cryosections of CAM tumor nodules. Representative images of Ki67 staining (magenta), DAPI-positive nuclei (blue), and merged channels are shown (left). Quantification of Ki67-positive cells as a percentage of total cells is shown on the right. Scale bars, 150 μm. (E) SRB assay of dose-dependent proliferation responses of U2OS cells to cisplatin under the indicated treatment conditions. Dose-response curves are shown in the upper panel, and calculated IC50 values are listed below. (F) Flow cytometric analysis of apoptosis using Annexin V-FITC/PI staining following cisplatin treatment. Representative dot plots are shown (top), with quantification of apoptotic cells shown on the bottom. (G) Kaplan-Meier survival analysis of TARGET-OS patients stratified according to combined TGFB1 and MDM2 expression levels. Patients with high TGFB1 and low MDM2 expression showed the most favorable survival outcome. Data are presented as mean ± SD. p values in (B, D, and F) were determined using one-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (Novoprotein, China) was diluted in complete medium to a final concentration of 5 ng/mL , and added directly to the culture medium as part of the experimental treatment.

Techniques: Membrane, Chick Chorioallantoic Membrane Assay, Stable Transfection, Expressing, Fluorescence, Imaging, Labeling, Immunofluorescence, Staining, Sulforhodamine B Assay

Journal: IBRO Neuroscience Reports

Article Title: TGF-β1 modulates PFC glial cell activation to facilitate exercise-induced analgesia in mice with spared nerve injury

doi: 10.1016/j.ibneur.2026.03.009

Figure Lengend Snippet: Exercise modulates TGF-β1 expression in the prefrontal cortex (PFC) of mice 24 days after spared nerve injury (SNI). (a) Representative Western blot images of TGF-β receptor I (TGF-βR1) and TGF-β1 in the PFC. Tissue lysates from all experimental groups (SHAM, SHAME, SNI, SNIE) and recombinant human TGF-β1 (non-reduced and reduced) were loaded on the same SDS–PAGE gel, transferred to a single membrane, and probed with the same TGF-β1 antibody under identical exposure conditions. The recombinant protein (250 ng per lane) served as a positive control to verify the molecular weights of the dimeric (25 kDa) and monomeric (12.5 kDa) forms of TGF-β1. GAPDH was used as the loading control. (b-d) Quantitative Western blot analyses of (b) TGF-βR1, (c) dimeric TGF-β1 (25 kDa), and (d) monomeric TGF-β1 (12.5 kDa) expression levels in tissue lysates. Data are presented as mean ± SEM (n = 3). ** P < 0.01 vs. SHAM group; ## P < 0.01 vs. SNI group.

Article Snippet: To validate the specificity of the TGF-β1 antibody, Recombinant human TGF-β1 protein (Catalog # 240-B, R&D Systems, USA) was used as a positive control.

Techniques: Expressing, Western Blot, Recombinant, SDS Page, Membrane, Positive Control, Control

Journal: IBRO Neuroscience Reports

Article Title: TGF-β1 modulates PFC glial cell activation to facilitate exercise-induced analgesia in mice with spared nerve injury

doi: 10.1016/j.ibneur.2026.03.009

Figure Lengend Snippet: At 24 d after SNI, mouse PFC astrocytes were activated and microglia were unchanged. (a)Western blotting analysis of changes in GFAP and Iba1 expression in PFC (n = 3); (b) Quantification of GFAP in PFC; (c) Quantification of Iba1 in PFC; (d) MFI representative images of GFAP in PFC; (e) MFI representative image of Iba1 in PFC; (f) Quantification of GFAP in PFC. Values represent mean ± SEM (Scale bar =75μm, 9 PFC sections from 3 mice per group); (g) Quantification of Iba1 in PFC. Values represent mean ±SEM (Scale bar = 75μm, 9 PFC sections from 3 mice per group). Values represent the mean ±SEM. * P < 0.05, ** P < 0.01, compared with SHAM group; # P < 0.05, ## P < 0.01, compared with SNI group, the difference was statistically significant; (h) Representative MFI images of changes in the colocalization of TGF-β1(red) and astrocytes (green) in the PFC; (i) Quantification of TGF-β1 and astrocytes in PFC. Values represent the mean ± SEM (Scale bar =100μm, nine PFC sections from three mice per group). * P < 0.05 versus the SHAM group; # P < 0.05 versus the SNI group.

Article Snippet: To validate the specificity of the TGF-β1 antibody, Recombinant human TGF-β1 protein (Catalog # 240-B, R&D Systems, USA) was used as a positive control.

Techniques: Western Blot, Expressing

Journal: IBRO Neuroscience Reports

Article Title: TGF-β1 modulates PFC glial cell activation to facilitate exercise-induced analgesia in mice with spared nerve injury

doi: 10.1016/j.ibneur.2026.03.009

Figure Lengend Snippet: TGF-βRI inhibition reverses exercise-induced analgesia and modulates glial activation in the PFC. (a, b) Time course of mechanical and cold hyperalgesia tests (n = 9). The green shading indicates the duration of the exercise intervention, and the green vertical lines denote the timing of intrathecal injections. Data are presented as mean ± SEM. ** P < 0.01 versus the SNIE group, # P < 0.05, ## P < 0.01 vs. SC group. (c) Representative Western blot images of TGF-βR1 and TGF-β1 in the PFC. Tissue lysates from SC and SA groups and recombinant human TGF-β1 (100 ng per lane) (non-reduced and reduced) were loaded on the same SDS–PAGE gel, transferred to a single membrane, and probed with the same TGF-β1 antibody in a single exposure without splicing. The recombinant protein served as a positive control to verify the molecular weights of the dimeric (25 kDa) and monomeric (12.5 kDa) forms of TGF-β1. GAPDH was used as the loading control. (d-f) Quantitative analysis of (d) TGF-βR1, (e) dimeric TGF-β1 (25 kDa), and (f) monomeric TGF-β1 (12.5 kDa) expression levels (n = 3). (g-i) Western blot analysis of glial markers. (g) Representative images of GFAP and Iba1 with GAPDH control. Quantitative analysis of (h) GFAP and (i) Iba1 expression levels (n = 3). (j, k) Representative immunofluorescence images showing the expression of (j) GFAP and (k) Iba1 in the PFC. Scale bar = 75 μm. (l, m) Quantification of the mean fluorescence intensity (MFI) for (l) GFAP and (m) Iba1 (n = 9 sections from 3 mice per group). Data in bar graphs are presented as mean ± SEM. * P < 0.05, ** P < 0.01 vs. SC group. SC: Spared nerve injury with exercise training followed by intrathecal (i.t.) injection of saline; SA: Spared nerve injury with exercise training followed by i.t. injection of the TGF-βRI inhibitor.

Article Snippet: To validate the specificity of the TGF-β1 antibody, Recombinant human TGF-β1 protein (Catalog # 240-B, R&D Systems, USA) was used as a positive control.

Techniques: Inhibition, Activation Assay, Western Blot, Recombinant, SDS Page, Membrane, Positive Control, Control, Expressing, Immunofluorescence, Fluorescence, Injection, Saline