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: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a Experimental design. b Representative bioluminescent image of pre-adoptive and post-adoptive C57BL/6 J mice receiving Ly108 + CX3CR1 - , Ly108 + CX3CR1 + , Ly108 - CX3CR1 + , Ly108 - CX3CR1 - cells transfer or without transfer ( n = 5 mice/group). c Quantification of ( b ). d Survival curve of tumor-bearing mice for indicated groups described in ( b ). Log-rank test for survival comparison. e Flow cytometry depicted the phenotype of adoptively transferred cells before and 14 days after transfer. All subsets shown were gated on CD8 + PD1 + CD44 + CD45.2 + CD45.1 - cells. f Representative bioluminescent image of TβRI-hWT and TβRI-hKO mice inoculated with TGFβ1 OE , TGFβ1 KD , or TGFβ1 CTL PAN02-Luc cells ( n = 6 mice/group). g Quantification of ( f ). Data are presented as the mean ± SD, and were analyzed with one-way ( g ) or two-way ( c ) ANOVA with Tukey’s multiple comparison test. Consistent results were observed across three biological replicates in ( b–g ). Source data are provided as a Source Data file.

Article Snippet: For in vitro knockdown of TGFβ1 in PAN02-luc cells, the custom-made GV654 lentiviral vectors encoding shRNAs with sequences targeting mouse TGFβ1 were purchased from GENECHEM (Shanghai, China).

Techniques: Comparison, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a Luminex showing the protein levels of inflammatory cytokines in tumor interstitial fluid of TβRI-hWT and TβRI-hKO mice ( n = 3 mice/group). Data are shown as a heatmap. b Experimental scheme for generating Gal-9 flox/flox Alb-Cre +/- (Gal9-hKO) mice. Gal-9 flox/flox Alb-Cre -/- and C57BL/6 J were used as Gal9-hWT mice. c Representative bioluminescent image of Gal9-hWT versus Gal9-hKO mice inoculated with TGFβ1 OE or TGFβ1 CTL -PAN02 cells in the portal vein on day 28 ( n = 6 mice/group). d Bioluminescence quantification of ( c ). e Statistical analysis depicting the percentages of CD39 + TOX + cells within CD8 + PD1 + CD44 + tumor-reactive T cells in the indicated groups. f Statistical analysis depicting the percentages of Tex subsets based on TCF1 and CX3CR1 within CD8 + PD1 + CD44 + tumor-reactive T cells. g statistical analysis depicting the TNF and IFNγ co-production by CD8 + CD44 + T subsets in the indicated groups upon ex vivo CD3/CD28 stimulation. h The diagram of experimental design. i Representative bioluminescent image of Gal9-hWT versus Gal9-hKO mice treated with IgG, αCD4, αCD8 or αPD-1( n = 5 mice/group). j Representative macroscopic image of ( i ). k Bioluminescence quantification of ( i ). l Statistical analysis depicting the percentages of Tex subsets based on Ly108 and CX3CR1 within CD8 + PD1 + CD44 + tumor-reactive T cells. Data are presented as the mean ± SD, and were analyzed with unpaired two-sided Student’s t test ( k ) or two-way ( d , e , f , g , l ) ANOVA with Tukey’s multiple comparison test. Consistent results were observed across three biological replicates in ( a , c–g , i–l ). Source data are provided as a Source Data file.

Article Snippet: For in vitro knockdown of TGFβ1 in PAN02-luc cells, the custom-made GV654 lentiviral vectors encoding shRNAs with sequences targeting mouse TGFβ1 were purchased from GENECHEM (Shanghai, China).

Techniques: Luminex, Ex Vivo, Comparison

Journal: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a Schematic of CP-LNP1-Gal, a hepatocyte-targeting nano-liposome for TGFβ signaling inhibition, labeled with DID for in vivo tracking. Created in BioRender. Zhong, S. (2025) https://BioRender.com/qphmhxl b Representative ex vivo fluorescence images of various organ at indicated time points. Green dashed line: Liver metastasis; White dashed line: Normal liver. c–g Immunofluorescence analysis of DID localization within the liver metastatic microenvironment. LM: Liver metastasis; L: Normal Liver. The border between the liver metastatic lesion and the normal liver tissue is demarcated by a white dashed line. Scale bar, 20 μm. c Representative images of immunofluorescence co-staining for DID (pink) with Alb (green). d Representative images of immunofluorescence co-staining for DID (pink) with CD31 (green). e Representative images of immunofluorescence co-staining for DID (pink) with Desmin. f Representative images of immunofluorescence co-staining for DID (pink) with F4/80 (green). g Quantitative co-localization analysis using Pearson’s correlation coefficient. h, i Immunofluorescence co-staining for DID (pink) and p-smad2 (red) with Alb (green) or F4/80 (green) in mouse liver metastases. LM: Liver metastasis; L: Normal Liver. The border between the liver metastatic lesion and the normal liver tissue is demarcated by a white dashed line. Scale bar, 30 μm. h Representative images (Left) of immunofluorescence co-staining for DID (pink) and p-smad2 (red) with Alb (green). Quantification of p-smad2 in Alb-positive hepatocytes. i Representative images (Left) of immunofluorescence co-staining for DID (pink) and p-smad2 (red) with F4/80 (green). Quantification of p-smad2 in F4/80 positive macrophages. Data are presented as the mean ± SD, and were analyzed with one-way ANOVA with Sidak’s multiple comparison test ( g ) or unpaired two-sided Student’s t test ( h , i ). Consistent results were observed across three biological replicates in ( b–i ). Source data are provided as a Source Data file.

Article Snippet: For in vitro knockdown of TGFβ1 in PAN02-luc cells, the custom-made GV654 lentiviral vectors encoding shRNAs with sequences targeting mouse TGFβ1 were purchased from GENECHEM (Shanghai, China).

Techniques: Inhibition, Labeling, In Vivo, Ex Vivo, Fluorescence, Immunofluorescence, Staining, Comparison