Journal: Science Advances

Article Title: TDRD3, a Tudor domain-containing protein, regulates Klf2 -dependent T reg differentiation and function to modulate immune tolerance

doi: 10.1126/sciadv.aea3960

Figure Lengend Snippet: ( A and B ) Immunoblot analysis of TDRD3 in CD4 + YFP + T regs (A) or CD4 + cells (B) isolated from the spleen of indicated mice. Right: qPCR analysis of Tdrd3 mRNA ( n ≥ 3). ( C ) Representative flow cytometric analysis (left two panels), percentage (third panel) and number (right panel) of T reg (Foxp3 + ) cells in thymocytes from indicated mice ( n ≥ 6). ( D and E ) Representative flow cytometric analysis (left two panels) and percentage (third panel) of Foxp3 + iT regs among CD4 + T cells from Foxp3 YFP-Cre or Tdrd3 fl/fl /Foxp3 YFP-Cre mice (D) or from Tdrd3 fl/fl and Tdrd3 fl/fl /CD4 Cre mice (E), polarized in the presence of TGF-β (5 ng/ml) for 48 hours ( n ≥ 6 per treatment cohort). Foxp3 mRNA levels in differentiated iT regs were detected by qPCR (right). ( F ) Representative flow cytometric analysis (two left panels) and percentage (right panel) of live cells among Foxp3 + iT regs differentiated from naïve CD4 + T cells of indicated mice ( n ≥ 6). ( G ) Representative flow cytometric analysis (two left panels) and percentage (right panel) of the proliferating Foxp3 YFP-Cre or Tdrd3 fl/fl /Foxp3 YFP-Cre CD4 + T cells in indicated peaks shown on left, labeled with CellTrace Violet (CTV) and polarized under T reg conditions for 48 hours ( n ≥ 6). Boxed area: cell population of interest. Data are from three independent experiments [(A), (B), (C), (E), (F), and (G), right panels; presented as means ±SEM] or are from one representative of three independent experiments [(A), (B), (C), (E), (F), and (G), left panels]. ** P < 0.01, *** P < 0.001, and **** P < 0.0005; ns, not significant (two-tailed Student’s t test).

Article Snippet: Recombinant TGF-β (#130-095-067) was from Miltenyi Biotec.

Techniques: Western Blot, Isolation, Labeling, Two Tailed Test

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