Journal: Frontiers in Pharmacology

Article Title: Licoricesaponin G2 ameliorates bleomycin-induced pulmonary fibrosis via targeting TNF-α signaling pathway and inhibiting the epithelial-mesenchymal transition

doi: 10.3389/fphar.2024.1437231

Figure Lengend Snippet: Effects of LG2 on cell viability in A549 and MRC5 cells. (A,B) Cytotoxicity of LG2 in A549 and MRC5 cells following 48-h treatment with various doses. (C,D) Impact of LG2 on cell viability in TGF-β1–induced A549 and MRC5 cells post 48-h treatment (n = 3).* p < 0.05, ** p < 0.005, *** p < 0.001, compared to the Ctrl group (TGF-β1, 5 ng/mL).

Article Snippet: Bleomycin (BLM, HY17565), pirfenidone (HYB0673), TGF-β1 (HY-151427) and Etanercept (HY-108847) were acquired from MedChemExpress, Princeton, United States.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Licoricesaponin G2 ameliorates bleomycin-induced pulmonary fibrosis via targeting TNF-α signaling pathway and inhibiting the epithelial-mesenchymal transition

doi: 10.3389/fphar.2024.1437231

Figure Lengend Snippet: Inhibition of TGF-β1–induced migration in A549 cells by LG2. (A) Effects of LG2 on enhanced migration ability of A549 cells induced by TGF-β1, determined via a transwell migration assay. (B) Quantitative analysis using ImageJ software. (C) IF image showing α-SMA distribution. (D) Quantitative analysis of α-SMA expression. (E) IF image showing Collagen I distribution. (F) Quantitative analysis of Collagen I expression (scale bar = 50 μm) (n = 3). * p < 0.05, ** p < 0.005, *** p < 0.001, compared to the Model group. # p < 0.05, ## p < 0.005, ### p < 0.001, compared to the Ctrl group.

Article Snippet: Bleomycin (BLM, HY17565), pirfenidone (HYB0673), TGF-β1 (HY-151427) and Etanercept (HY-108847) were acquired from MedChemExpress, Princeton, United States.

Techniques: Inhibition, Migration, Transwell Migration Assay, Software, Expressing

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: ADAMTS1 levels were increased in the serum of patients with myocardial fibrosis, in the hearts of mice after CFPMI and in cardiac fibroblasts stimulated with fibrotic factors. A . ADAMTS1 levels in clinical serum samples were evaluated via ELISA. N = 30. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. B . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. C . Survival rate of mice in each group. D . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. E and F . ADAMTS1 expression in mouse heart tissue was determined through Western blot and IHC. Human cardiac fibroblasts were treated with 10 ng/mL TGF-β1 or 0.1 μM Ang II for 12, 24, or 48 h to construct in vitro models. The cells were divided into four groups: the Control, TGF-β1/Ang II (12 h), TGF-β1/Ang II (24 h), and TGF-β1/Ang II (48 h) groups. G and H . Western blot detection of the expression of ADAMTS1, Collagen I and FN. I. The expression levels of ADAMTS1, Collagen I and FN were measured via qRT‒PCR. After the optimal time point (48 h) was determined, the mice were further divided into the control, TGF-β1 and Ang II groups. J . ADAMTS1 expression in cells was assessed by IF. Human cardiac fibroblasts were treated with 10 ng/mL TGF-β1 or 0.1 μM Ang II for 15, 30, 60, 90, and 120 min. K. qRT-PCR detection of the mRNA expression of ADAMTS1, Collagen I and FN. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Construct, Staining, Expressing, Western Blot, In Vitro, Control, Quantitative RT-PCR

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: SMAD2 regulated ADAMTS1 expression in human and mouse cardiac fibroblasts induced by TGF-β1. We used 10 ng/mL TGF-β1 to induce human and mouse cardiac fibroblasts for 15, 30, 60, 90, and 120 min. A and B . Western blot analysis of the expression of SMAD2, p-SMAD2 and ADAMTS1. Human and mouse cardiac fibroblasts were treated with 10 ng/mL TGF-β1 for 48 h, and the cells were divided into Control and TGF-β1 groups. C . Human and mouse cardiac fibroblasts were induced with 10 ng/mL TGF-β1 for 12 h, 24 h, 48 h, and 72 h. qRT‒PCR was performed to detect the mRNA levels of ADAMTS1, Collagen I, and FN. D . IF staining was conducted to determine the expression of p-SMAD2 and ADAMTS1 at 48 h. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. E . p-SMAD2 expression in mouse heart tissue was measured by IHC. F . ChIP verification of the interaction between SMAD2 and ADAMTS1. Then, we interfered with SMAD2 expression. The groups were the si-NC, si-SMAD2-1, and si-SMAD2-2 groups. G . SMAD2 expression was assessed via qRT‒PCR. After the best si-SMAD2 was selected, the cells were further divided into the si-NC, TGF-β1 (48 h), si-SMAD2, and TGF-β1 + si-SMAD2 groups. H . Western blot detection of ADAMTS1, Collagen I, and FN expression in cells. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control, Staining, Construct

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: Overexpression of ADAMTS1 enhanced the production of collagen fiber proteins in human and mouse cardiac fibroblasts induced by TGF-β1. We overexpressed ADAMTS1 and divided the cells into the oe-NC, TGF-β1 (48 h), oe-ADAMTS1, and TGF-β1 + oe-ADAMTS1 groups. A . Western blot detection of ADAMTS1, Collagen I, FN, and α-SMA expression. B-D . IF staining of Collagen I and FN expression. Additionally, we interfered with ADAMTS1; the cells were grouped as follows: si-NC, si-ADAMTS1-1, and si-ADAMTS1-2. E . ADAMTS1 expression was determined through qRT‒PCR. After the best si-ADAMTS1 was selected, the cells were further divided into the si-NC, TGF-β1 (48 h), si-ADAMTS1, and TGF-β1 + si-ADAMTS1 groups. F and G . Western blot detection of ADAMTS1, Collagen I, FN, and α-SMA expression. H and I . IF staining of Collagen I and FN expression. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Over Expression, Western Blot, Expressing, Staining

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: Knockdown of ADAMTS1 alleviated TGF-β1-induced fibrosis by downregulating HDAC6 protein expression. A CFPMI mouse model was constructed, and the mice were randomly divided into a Sham group and a CFPMI group, with 6 mice/group. A . HDAC6 expression in mouse heart tissue was determined through IHC. After the best si-ADAMTS1 was selected, the cells were further divided into the si-NC and si-ADAMTS1 groups. B . ADAMTS1 and HDAC6 expression was assessed via qRT‒PCR. C . Western blot analysis of ADAMTS1 and HDAC6 expression. Furthermore, we overexpressed ADAMTS1 and divided the cells into the oe-NC, TGF-β1, oe-ADAMTS1, and TGF-β1 + oe-ADAMTS1 groups. D . Western blot detection of HDAC6 expression. After the si-ADAMTS1 group was selected, the cells were further divided into the si-NC, TGF-β1, si-ADAMTS1, and TGF-β1 + si-ADAMTS1 groups. E. Western blot detection of HDAC6 expression. Moreover, cells were treated with the HDAC6 inhibitor ACY1215 (5 μM) for 48 h and then divided into the si-NC, si-ADAMTS1, si-NC + ACY1215, and si-ADAMTS1 + ACY1215 groups. F. Western blot detection of TGF-β1, Collagen I, FN, and ADAMTS1 expression. Subsequently, experiments were performed in human and mouse cardiac fibroblasts with ADAMTS1 overexpression followed by treatment with the HDAC6 inhibitor ACY1215. G. Western blot analysis was conducted to detect the expression levels of ADAMTS1 and HDAC6. H. Western blot analysis was performed to determine the expression levels of TGF-β1, Collagen I, and FN. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Knockdown, Expressing, Construct, Western Blot, Over Expression

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: ADAMTS1 interacted with HDAC6 during fibrosis. A . Co-IP verification of the interaction of ADAMTS1 with HDAC6, with ADAMTS1 as the bait protein. In the experiment, we first captured the ADAMTS1 protein using a specific antibody and then used Co-IP technology to detect its interaction with HDAC6. B . Co-IP verification of the interaction of HDAC6 with ADAMTS1, with HDAC6 as the bait protein. In the experiment, we captured the HDAC6 protein using a specific antibody and then used Co-IP technology to detect its interaction with ADAMTS1. We used 10 ng/mL TGF-β1 to induce human and mouse cardiac fibroblasts for 48 h, and the cells were further divided into control and TGF-β1 groups. C . IF staining was performed to evaluate the colocalization of ADAMTS1 and HDAC6 in TGF-β1-treated human and mouse cardiac fibroblasts. D . Changes in the level of ubiquitinated HDAC6 protein in human and mouse cardiac fibroblasts transfected with oe-ADAMTS1/si-ADAMTS1 or oe-NC/si-NC in the presence of 10 μM MG132. E. In si-ADAMTS1-treated human and mouse cardiac fibroblasts, MG132 (10 μM) was added to detect the ubiquitination levels of ADAMTS1 protein. F. Human and mouse cardiac fibroblasts were treated with TGF-β1 and subjected to SMAD2 knockdown to determine the ubiquitination levels of ADAMTS1 protein. N = 3. *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Co-Immunoprecipitation Assay, Control, Staining, Transfection, Ubiquitin Proteomics, Knockdown

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: AAV-shRNA-ADAMTS1 treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. The mice were randomly divided into the Sham, CFPMI, and CFPMI + sh-ADAMTS1 groups. A . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. B . Survival rate of mice in each group. C . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. D . HDAC6 expression was determined through qRT‒PCR. E. Western blot analysis of the expression of TGF-β1, SMAD2, and p-SMAD2 in mouse heart tissue. F. Western blot detection of Collagen I, and FN expression. N = 3. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: shRNA, Staining, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: AAV-shRNA-HDAC6 transfection combined with ADAMTS1 inhibitor treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. The mice were randomly divided into the Sham, CFPMI, CFPMI + anti-ADAMTS1, CFPMI + sh-HDAC6, and CFPMI + anti-ADAMTS1 + sh-HDAC6 groups. A . Echocardiographic results of different treatment groups, with measurements of ejection fraction (EF) and fractional shortening (FS). N = 6. B . Survival rate of mice in each group. C and D . HE and picrosirius red staining of collagen deposition in mouse cardiac tissues, with quantification of the Collagen Volume Fraction. E. HDAC6 expression was determined through qRT‒PCR. F. Western blot analysis of the expression of TGF-β1, SMAD2, and p-SMAD2 in mouse heart tissue. G. Western blot detection of Collagen I and FN expression. N = 3. ** P < 0.01, *** P < 0.001

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: shRNA, Transfection, Staining, Expressing, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Targeting ADAMTS1/HDAC6 alleviates TGF-β1/SMAD2-associated cardiac fibrosis in cardiac fibrosis post-myocardial infarction

doi: 10.1007/s10565-026-10159-2

Figure Lengend Snippet: TGF-β1/SMAD2 regulated ADAMTS1 by mediating CFPMI through HDAC6 ubiquitination

Article Snippet: The blocked membranes were incubated overnight at 4 °C with the following primary antibodies: ADAMTS1 (ab236497, 1:2000, Abcam), Collagen I (ab270993, 1:1000, Abcam), FN ( AWA00143 , 1:1000, Abiowell), SMAD2 (12,570–1-AP, 1:5000, Proteintech), p-SMAD2 (ab280888, 1:1000, Abcam), α-SMA (ab5694, 1 μg/ml, Abcam), HDAC6 (128,341-AP, 1:1000, Proteintech), TGF-β1 ( AWA00083 , 1:1000, Abiowell), and GAPDH (10,494–1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-β Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

doi: 10.1371/journal.pone.0083846

Figure Lengend Snippet: In gene therapy, treated rats with renal transplants TGF-β were evaluated by immunohistochemistry. Active TGF-β was either detected directly by using an antibody recognizing active TGF-β (A) or indirectly by evaluation of phosphorylation of the TGF-β signaling molecule smad2/3 (B) or expression of the TGF-β downstream target PAI-1 within the glomeruli (C) or the cortex (D). Representative microphotographs of PAI-1 staining in renal grafts treated with control (E) and TSP-2 overexpressing plasmid (F) are shown. Total TGF-β1 (G) and TGF-β2 (H) was similar in both groups. Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,05.

Article Snippet: The TGF-β system was studied using antibodies to TGF-β1 (rabbit anti-human TGF-β1, Santa Cruz Biotechnology Inc. ); TGF-β2 (rabbit anti-human TGF-β2, Santa Cruz ); active TGF-β1 (chicken anti-human active TGF-β1, (R&D systems, Germany , , Phospho-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435, Santa Cruz ) or PAI-1, a rabbit polyclonal to human plasminogen activator inhibitor-1 (Santa Cruz).

Techniques: Immunohistochemistry, Phospho-proteomics, Expressing, Staining, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-β Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

doi: 10.1371/journal.pone.0083846

Figure Lengend Snippet: Representative microphotographs from immunohistological staining of kidney grafts for active TGF-β (A, control plasmid; B, TSP-2 therapy, brown cytosolic staining), P-smad 2/3 (C, control plasmid; D, TSP-2 therapy, brown nuclear staining), PAI-1 (E, control plasmid; F, TSP-2 therapy, brown staining), fibronectin (G, control plasmid; H, TSP-2 therapy, brown staining) and alpha-smooth muscle actin (I, control plasmid; J, TSP-2 therapy, brown staining) are shown.

Article Snippet: The TGF-β system was studied using antibodies to TGF-β1 (rabbit anti-human TGF-β1, Santa Cruz Biotechnology Inc. ); TGF-β2 (rabbit anti-human TGF-β2, Santa Cruz ); active TGF-β1 (chicken anti-human active TGF-β1, (R&D systems, Germany , , Phospho-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435, Santa Cruz ) or PAI-1, a rabbit polyclonal to human plasminogen activator inhibitor-1 (Santa Cruz).

Techniques: Staining, Control, Plasmid Preparation

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: Journal of Clinical Investigation

Article Title: Age-associated callus senescent cells produce TGF-β1 that inhibits fracture healing in aged mice

doi: 10.1172/jci148073

Figure Lengend Snippet: Figure 8. TGF-β neutralization enhances fracture healing in aged mice. 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 concen- tration 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: Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Injection, Micro-CT, Staining, Software, Flow Cytometry