Journal: Stem Cell Research & Therapy

Article Title: uPAR deficiency triggers TGFβ1-mediated fibrotic remodeling in a cardiac perivascular-like microenvironment

doi: 10.1186/s13287-026-04923-8

Figure Lengend Snippet: uPAR deficiency promotes TGFβ1 activation, uPA nuclear accumulation and SNAIL upregulation. A Protein and gene expression levels of transforming growth factor β1 (TGFβ1) in Wt and uPAR-/- mCSs, * p < 0.05, N = 3–4. B Protein levels of single-chain urokinase (sc-uPA) and two-chain urokinase (tc-uPA) in Wt and uPAR-/- mCSs, ** p < 0.01, N = 4. C uPA nuclear accumulation (percent of uPA-positive nuclei) within Wt and uPAR-/- mCSs, * p < 0.05, N = 3. D Confocal images of intracellular uPA staining (green) of Wt and uPAR-/- mCS cryosections. Nuclei were stained with DAPI. White segmented arrows indicate the direction of fluorescence intensity profiling presented on panel “E”. Straight arrows indicate colocalization of uPA and DAPI. E Intensity profile plots of the uPA and nuclei/DAPI fluorescence signal. Arrows indicate overlay of fluorescence signals. F Nuclear uPA expression (mean fluorescence intensity per nuclei) within Wt and uPAR-/- mCSs, * p < 0.05, N = 3. G Protein levels of SNAIL and TWIST1 in Wt and uPAR-/- mCSs, **** p < 0.0001, N = 4. H Representative images of western blot results for panels “A”, “B” and “G”. Full-length blots/gels are presented in Supplementary Figure S7. I Confocal images of double-staining for CD31 (red) and α smooth-muscle actin (SMA, green) in Wt and uPAR-/- mouse cardiospheres (mCSs). Nuclei were stained with DAPI. White arrows indicate colocalization of CD31 and SMA. J Intensity profile plots of the CD31 and SMA fluorescence signal. Arrows indicate overlay of fluorescence signals

Article Snippet: To analyze transforming growth factor beta 1 (TGFβ1)-induced signaling activation, cells were treated with 10 ng/mL mouse TGFβ1 (PKSM041167, Elabscience) in DMEM low glucose (Servicebio) for 30 min. Activation of SMAD2/SMAD3 and Akt pathways was assessed by immunoblotting using phospho/total protein ratios.

Techniques: Activation Assay, Gene Expression, Staining, Fluorescence, Expressing, Western Blot, Double Staining

Journal: Stem Cell Research & Therapy

Article Title: uPAR deficiency triggers TGFβ1-mediated fibrotic remodeling in a cardiac perivascular-like microenvironment

doi: 10.1186/s13287-026-04923-8

Figure Lengend Snippet: Plaur knockout in fibroblasts resulted in enhanced ECM deposition and TGFβ1 activation. A Schematic representation of Plaur knockout in fibroblasts using CRISPR/Cas9 system with three single guide RNAs (sgRNA). The illustration was created with Servier Medical Art, licensed under CC-BY 4.0. B Flow cytometry analysis of Wt, Scrambled (control plasmid) and Plaur knockout ( Plaur KO) fibroblasts stained with anti-uPAR antibodies (red), isotype IgG (green), or unstained (blue). C Plaur gene expression in fibroblasts quantified by qPCR, ** p < 0.01, *** p < 0.001, **** p < 0.0001, N = 3. D Analysis of surface uPAR expression on fibroblasts by flow cytometry. Data are presented as median fluorescence intensity (MFI), *** p < 0.001, **** p < 0.0001, N = 3. E Representative images of western blot results for panels “F” and “G”. Full-length blots/gels are presented in Supplementary Figure S8. F Protein levels of COL I (pro-collagen I and mature collagen I α-chain), FN, and αSMA measured in fibroblasts, * p < 0.05, ** p < 0.01, N = 3. G Protein levels of latent and active (dimer and monomer) forms of TGFβ1 in fibroblasts, * p < 0.05, ** p < 0.01, N = 3. H Tgfb1 gene expression in fibroblasts, * p < 0.05, ** p < 0.01, N = 3

Article Snippet: To analyze transforming growth factor beta 1 (TGFβ1)-induced signaling activation, cells were treated with 10 ng/mL mouse TGFβ1 (PKSM041167, Elabscience) in DMEM low glucose (Servicebio) for 30 min. Activation of SMAD2/SMAD3 and Akt pathways was assessed by immunoblotting using phospho/total protein ratios.

Techniques: Knock-Out, Activation Assay, CRISPR, Flow Cytometry, Control, Plasmid Preparation, Staining, Gene Expression, Expressing, Fluorescence, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: uPAR deficiency triggers TGFβ1-mediated fibrotic remodeling in a cardiac perivascular-like microenvironment

doi: 10.1186/s13287-026-04923-8

Figure Lengend Snippet: TGFβ1 further enhances Akt phosphorylation and ECM synthesis in Plaur knockout fibroblasts. A Schematic representation of the experimental design for assessing TGFβ1 effects on Plaur knockout ( Plaur KO) fibroblasts. B Analysis of SMAD2/SMAD3 and Akt phosphorylation in Scrambled and Plaur KO fibroblasts following 30 min TGFβ1 stimulation. Phospho-protein levels were normalized to total proteins levels, * p < 0.05, ** p < 0.01, N = 3. C Representative images of western blot results for panel “B”. Full-length blots/gels are presented in Supplementary Figure S9. D Immunofluorescence images of Wt, Scrambled and Plaur KO fibroblasts after 48-hour TGFβ1 stimulation stained for collagen I (COL I, green) and EDA-fibronectin (EDA-FN, red). Nuclei were stained with DAPI. E Protein levels of COL I (pro-collagen I and mature collagen I α-chain), FN, and αSMA measured in fibroblasts after 48-hour TGFβ1 stimulation, * p < 0.05, N = 3. F Representative images of western blot results for panel “E”. Full-length blots/gels are presented in Supplementary Figure S10. G Schematic summary illustrating that uPAR deficiency in fibroblasts promotes TGFβ1 activation, enhances TGFβ1-dependent signal transduction, and stimulates ECM synthesis. “A” and “G” were created with Servier Medical Art, licensed under CC-BY 4.0

Article Snippet: To analyze transforming growth factor beta 1 (TGFβ1)-induced signaling activation, cells were treated with 10 ng/mL mouse TGFβ1 (PKSM041167, Elabscience) in DMEM low glucose (Servicebio) for 30 min. Activation of SMAD2/SMAD3 and Akt pathways was assessed by immunoblotting using phospho/total protein ratios.

Techniques: Phospho-proteomics, Knock-Out, Western Blot, Immunofluorescence, Staining, Activation Assay, Transduction

Journal: Stem Cell Research & Therapy

Article Title: uPAR deficiency triggers TGFβ1-mediated fibrotic remodeling in a cardiac perivascular-like microenvironment

doi: 10.1186/s13287-026-04923-8

Figure Lengend Snippet: Fibrotic remodeling of the cardiac perivascular microenvironment induced by uPAR deficiency. uPAR: urokinase receptor; uPA: urokinase; EC: endothelial cell; SM: smooth muscle; EndMT: endothelial-to-mesenchymal transition; ECM: extracellular matrix; COL: collagen; TGFβ1: transforming growth factor β1; Created with Servier Medical Art, licensed under CC-BY 4.0

Article Snippet: To analyze transforming growth factor beta 1 (TGFβ1)-induced signaling activation, cells were treated with 10 ng/mL mouse TGFβ1 (PKSM041167, Elabscience) in DMEM low glucose (Servicebio) for 30 min. Activation of SMAD2/SMAD3 and Akt pathways was assessed by immunoblotting using phospho/total protein ratios.

Techniques:

Journal: International Journal of Oral Science

Article Title: PTHrP promotes subchondral bone formation in TMJ-OA

doi: 10.1038/s41368-022-00189-x

Figure Lengend Snippet: PTH promoted the osteoblastic differentiation of SMSCs. a Osteogenic induction cells (7 d) were stained by Alkaline phosphatase (ALP) staining and Alizarin Red S staining (ARS) at 14 d, which revealed the radically increased osteogenesis of SMSCs upon PTH treatment. b – d Real-time RT–PCR analysis shows osteogenic-related gene expression ( Sp7, Runx2, Bglap) at 7 d. * P < 0.05, *** P < 0.005. e Osteogenic-related protein (OCN) expression at 7 d with PTH treatment. f Western blot analysed downstream factors of the PTH-PTH1R and TGFβ pathways in SMSCs after PTH treatment for 7 d of osteogenesis. * P < 0.05, ** P < 0.01, *** P < 0.005. n = 3

Article Snippet: Primary antibodies matrix metallopeptidase 13 (MMP13, Abcam, Cambridge, UK; 1:200, ab3208), osterix (OSX) (Abcam, 1:200, ab22552), osteocalcin (OCN) (Takara Bio Inc., Shiga, Japan; 1:200, M137); a TGFβ pathway-specific antibody against p-Smad2/3 (Santa Cruz Biotechnology, 1:100, sc-11769); and antibody against PTH1R (Abcam, 1:200, ab15750).

Techniques: Staining, Quantitative RT-PCR, Gene Expression, Expressing, Western Blot

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A ) Representative images of collagen invasion of spheroids composed of HPMCs or TGF-β1–stimulated HPMCs. Scale bars, 200 μm. ( B ) The bar graph showing TGF-β1–stimulated HPMCs showed a higher invasion ability into the collagen layer. ( C ) Images of migration or invasion cells using the Transwell assay. Scale bar, 200 μm. ( D ) Linear plots showing that TGF-β1–stimulated HPMCs had a higher migration and invasion ability compared with the control HPMCs. ( E ) Bar graph showing the concentration of TGF-β1 in the supernatant in EOC cells when inhibiting TGF-β1 with siRNA. ( F ) Representative images of spheroid collagen invasion. Scale bars, 100 μm. ( G ) Bar graphs showing that inhibition of TGF-β1 by siRNA in OV90 cells or the TGF-β1 receptor blocker in HPMCs reduced the invasion ability of ACMSs compared with the control. ( H and I ) Differences in the spheroids in ascites when mice were injected with OV90 (green) and HPMCs (red). The number of spheroids was significantly decreased when OV90 cells were treated with siRNA for TGF-β1 than those with si-control. Scale bars, 100 μm. ( J and K ) Representative images of metastases on the omentum and bar graph showing the metastasis area on the omentum. Scale bars, 1000 μm. ( L ) Bar graph showing the TGF-β1 concentration in the culture supernatant in sh-control– or TGF-β1–transduced OV90 cells. ( M and N ) Representative images and bar graph showing the omental metastatic area ( n = 8). Scale bars, 1000 μm. ( O and P ) Representative images of confocal imaging of the omental micrometastasis area and bar graph showing the invasion depth of mesothelial cells from the metastatic border. Scale bars, 100 μm. ( Q and R ) Representative images and bar graph showing that both the number and size of spheroids in ascites were significantly decreased in mice injected with sh-TGF-β1 no. 1 or 2 O90 cells compared with sh-control mice. Scale bars, 100 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: As none of these siRNAs achieved significant mRNA suppression in EOC cells, we used a commercially available siRNA mix (Santa Cruz Biotechnology, sc-270322) for subsequent experiments (no. 4).

Techniques: Migration, Transwell Assay, Control, Concentration Assay, Inhibition, Injection, Imaging

Journal: The Journal of Clinical Investigation

Article Title: Age-associated callus senescent cells produce TGF- β 1 that inhibits fracture healing in aged mice

doi: 10.1172/JCI148073

Figure Lengend Snippet: Young and aged mice were sacrificed on 10 dpf. ( A ) Heatmap of the expression of SASP factors in callus as determined by qPCR. n = 3. ( B ) Expression of SASP factors in callus and nonfractured bone was assessed by qPCR. n = 4. Two-way ANOVA followed by Tukey’s post hoc test. ( C ) p16 + SCs isolated from young and aged callus. Expression of SASP factors examined by qPCR. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( D ) p16 + , TGF-β1 + , and p16 + TGF-β1 + cells were identified by flow cytometry. ( E ) Fold changes in p16 + , TGF-β1 + , and p16 + TGF-β1 + cell percentages in callus and nonfractured tibiae of aged versus young mice. n = 4–6. * P < 0.05, for callus versus bone, by unpaired, 2-tailed Student’s t test. ( F ) Percentage of p16 + TGF-β1 + cells in callus and nonfractured tibiae. * P < 0.05; ^ P < 0.05; # P < 0.05, for aged versus young, by 2-way ANOVA followed by Tukey’s post hoc test. ( G ) Expression of TGF-β1 in callus tissues following D+Q treatment by as determined by Western blotting. ( H ) CaMPCs were treated for 2 days with CM from young or aged callus with or without TGF-β–neutralizing Ab or IgG. Cell growth and proliferation were assessed by methylene blue staining or a BrdU incorporation assay. n = 4 wells. * P < 0.05, for IgG versus anti–TGF-β Ab; # P < 0.05, for aged anti–TGF-β Ab versus young IgG, by 2-way ANOVA followed by Tukey’s post hoc test. ( I ) Concentration of active TGF-β1 in CM from young and aged callus cultures measured by ELISA. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( J ) Callus pieces were harvested from young and aged mice and infected with Tgfb1 or scrambled (Ctl) shRNA lentivirus. CM was collected. The expression of Tgfb1 was measured by qPCR. CaMPCs were treated with the CM. Cell growth was assessed using a CCK8 kit. n = 3 wells. * P < 0.05, for control versus Tgfb1 shRNA, by 2-way ANOVA followed by Tukey’s post hoc test. The experiment was repeated once.

Article Snippet: The shRNA lentiviral particles targeting Tgfb1 (sc-37192-V) and a scrambled sequence (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Isolation, Flow Cytometry, Western Blot, Staining, BrdU Incorporation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, shRNA, Control

Journal: The Journal of Clinical Investigation

Article Title: Age-associated callus senescent cells produce TGF- β 1 that inhibits fracture healing in aged mice

doi: 10.1172/JCI148073

Figure Lengend Snippet: Young and aged mice underwent tibial fracture surgery. ( A ) The expression of Tgfb1 in fracture callus at indicated time points was measured by qPCR. n = 3. Relative mRNA expression is the fold-change versus young mice as 1. ( B ) The concentration of active TGF-β1 protein in fracture callus at indicated time points was measured by ELISA. n = 4. * P < 0.05, for aged versus young; # P < 0.05, for young versus young 0 dpf; ^ P < 0.05, for aged versus aged 0 dpf, by 2-way ANOVA followed by Tukey’s post hoc test ( A and B ). ( C ) Outline of the experimental design. Aged mice were given 2 μg in 10 μL TGF-β Ab, 1D11, or isotype IgG vehicle by intra-callus injection on 1, 3, 5, and 7 dpf and sacrificed on 10 dpf ( D – F and H – J ) or 28 dpf ( G ). n = 4–5. ( D ) Callus volume was measured by micro-CT. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( E ) Representative images of ABH-stained sections showing more woven bone and callus areas in the anti–TGF-β Ab–treated mice. Scale bar: 1 mm. ( F ) Woven bone and cartilage areas were analyzed using Visiopharm software. ( G ) Bone stiffness, strength and toughness were assessed by biomechanical testing at 28 dpf. ( H ) The percentage and number of MPCs identified as CD45 – CD31 – CD105 + cells in fracture callus were determined by flow cytometry. ( I ) Representative paraffin sections of callus immunostained with anti-Ki67 Ab to detect proliferating cells (arrowheads). External callus is indicated by the dashed lines. Scale bars: 500 μm. Original magnification, ×4 (enlarged insets). ( J ) The percentage of Ki67 + cells was quantified by ImageJ. * P < 0.05, by unpaired, 2-tailed Student’s t test ( F , G , H , and J ).

Article Snippet: The shRNA lentiviral particles targeting Tgfb1 (sc-37192-V) and a scrambled sequence (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Injection, Micro-CT, Staining, Software, Flow Cytometry