human adamts1  (R&D Systems)

Journal: Advanced Science

Article Title: Adamts1 Exacerbates Post‐Myocardial Infarction Scar Formation via Mechanosensing of Integrin α8

doi: 10.1002/advs.202504138

Figure Lengend Snippet: Adamts1 is upregulated in ECs post‐MI. A) Transcript level of ADAMTS1 at different time points after myocardial infarction (MI) ( n = 6 per group). 3 days: F (2,15) = 88.95, p = 4.79e‐009, 7 days: F (2, 15) = 191.1, p = 2.13e‐011, 14 days: F (2, 15) = 62.55, p = 5.28e‐008. 28 days: F (2, 15) = 160.8, p = 7.36e‐011. B) Western blot analysis of Adamts1 protein expression in mice with sham or MI surgery [ n = 6 per group, F (2,15) = 26.12, p = 1.30e‐005], as well as healthy donors ( n = 2) and patients with ischemic HF ( n = 4, t = 3.038). C) Immunofluorescence (IF) staining showing the expression and localization of Adamts1 in cardiac tissue of mice 2 weeks post‐MI. D) In situ immunofluorescence (ISI) staining demonstrating co‐localization of Adamts1 with endothelial cells (ECs) (CD31, EC biomarker), with quantitative analysis of Adamts1/CD31 co‐localization coefficient in Sham and MI‐2W groups ( n = 6 per group). E) Transcript level of ADAMTS1 in ECs under normoxic and hypoxic conditions ( n = 6, t = 4.796). Statistical analysis was performed using one‐way ANOVA followed by Bonferroni post hoc test (A,B) and unpaired t tests (B,E). Data are presented as the mean ± SD. Adamts1, a disintegrin and metalloproteinase with thrombospondin motif 1; IA, infarct area; RA, remote infarct area; HF, heart failure; WGA, wheat germ agglutinin; MI, myocardial infarction.

Article Snippet: To investigate ADAMTS1‐mediated ITGα8 activation, two experiments were conducted: (1) NRCFs were treated with recombinant human ADAMTS1 (100 ng mL −1 , R&D Systems) or PBS control, followed by co‐immunoprecipitation and IF staining to assess ADAMTS1‐ITGα8 interaction and activation. (2) NRCFs were cultured on 1%, 2%, or 3% hydrogels for 24 h to evaluate ITGα8 activation in response to altered substrate stiffness using IF staining.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, In Situ, Biomarker Discovery

Journal: Advanced Science

Article Title: Adamts1 Exacerbates Post‐Myocardial Infarction Scar Formation via Mechanosensing of Integrin α8

doi: 10.1002/advs.202504138

Figure Lengend Snippet: Overexpression of Adamts1 aggravates cardiac dysfunction and scar formation post‐MI. A,B) Representative M‐mode echocardiography images and quantitative analysis of cardiac function in VEC Tie and ADAMTS1 Tie mice ( n = 6 per group). C) Representative B‐mode echocardiographic images showing ventricular wall expansion and motion in ADAMTS1 Tie and VEC Tie mice. D) Echocardiographic quantification of end‐systolic volume (ESV), end‐systolic diameter (ESD), stroke volume, and cardiac output at 2 weeks post‐MI in ADAMTS1 Tie and VEC Tie mice ( n = 6 per group). E) Representative 3D speckle tracking images demonstrating endocardial wall displacement. F) Quantitative analysis of radial strain and longitudinal strain in endocardial motility ( n = 6 per group). G,H) Masson's trichrome staining and quantitative analysis of infarct area (IA) from ligation site to apex in VEC Tie and ADAMTS1 Tie mice ( n = 5 per group). t (Section ) = 5.743, t (Section ) = 8.963, t (Section ) = 5.710, t (Section ) = 5.655, t (Section 5) = 5.410. Statistical analysis was performed using multiple unpaired t tests with two‐stage step‐up method of Benjamini, Krieger, and Yekutieli correction H, and two‐way ANOVA followed by Bonferroni post hoc test in B, D, and F. Data are presented as mean ± SD. Adamts1, a disintegrin and metalloproteinase with thrombospondin motif 1; VEC, vector; MI, myocardial infarction; vol, volume; dia, diameter; Endo, endocardium; Pk, peak; LV, left ventricular.

Article Snippet: To investigate ADAMTS1‐mediated ITGα8 activation, two experiments were conducted: (1) NRCFs were treated with recombinant human ADAMTS1 (100 ng mL −1 , R&D Systems) or PBS control, followed by co‐immunoprecipitation and IF staining to assess ADAMTS1‐ITGα8 interaction and activation. (2) NRCFs were cultured on 1%, 2%, or 3% hydrogels for 24 h to evaluate ITGα8 activation in response to altered substrate stiffness using IF staining.

Techniques: Over Expression, Staining, Ligation, Plasmid Preparation

Journal: Advanced Science

Article Title: Adamts1 Exacerbates Post‐Myocardial Infarction Scar Formation via Mechanosensing of Integrin α8

doi: 10.1002/advs.202504138

Figure Lengend Snippet: Overexpression of Adamts1 increases the accumulation of extracellular matrix (ECM) and tunes the mechanical structure of infarct scar. A) Immunofluorescence (IF) staining showing cardiac fibroblast activation and proliferation, and ECM accumulation in infarct scar from ADAMTS1 Tie and VEC Tie mice. B) Quantitative analysis of collagen I area, BrdU + cardiac fibroblasts, and α‐SMA + area ( n = 6, 4 fields per replicate). Statistical significance; t (ColI area) = 3.887, t (Brdu + CFs) = 2.432; t (α‐SMA) = 6.886. C) Representative transmission electron microscopy (TEM) images showing scar fiber organization in ADAMTS1 Tie and VEC Tie mice. D) Histogram showing distribution of collagen fiber diameters and quantitative analysis of fiber diameter ( n = 3 per group, t = 3.673). E) Schematic diagram of Young's modulus detection by atomic force microscopy (AFM) and representative force–distance curves. F) Quantitative analysis of Young's modulus in scar tissue ( n = 3 per group, t = 6.027). Statistical analysis was performed using unpaired t tests. Data are presented as the mean ± SD. Adamts1, a disintegrin and metalloproteinase with thrombospondin motif 1; Col, collagen; VCAN, versican; YM, Young's modulus.

Article Snippet: To investigate ADAMTS1‐mediated ITGα8 activation, two experiments were conducted: (1) NRCFs were treated with recombinant human ADAMTS1 (100 ng mL −1 , R&D Systems) or PBS control, followed by co‐immunoprecipitation and IF staining to assess ADAMTS1‐ITGα8 interaction and activation. (2) NRCFs were cultured on 1%, 2%, or 3% hydrogels for 24 h to evaluate ITGα8 activation in response to altered substrate stiffness using IF staining.

Techniques: Over Expression, Immunofluorescence, Staining, Activation Assay, Transmission Assay, Electron Microscopy, Microscopy

Journal: Advanced Science

Article Title: Adamts1 Exacerbates Post‐Myocardial Infarction Scar Formation via Mechanosensing of Integrin α8

doi: 10.1002/advs.202504138

Figure Lengend Snippet: Adamts1 deficiency improves cardiac function and reduces scar formation Post‐MI. A) Schematic illustration of Adamts1 conditional knockout (CKO) mouse generation and experimental timeline. Tamoxifen (50 mg kg −1 ) was administered 1 week before and after MI surgery. B) Representative M‐mode echocardiographic images and 3D speckle tracking analysis showing wall displacement in ADAMTS1 CKO and ADAMTS1 fl/fl mice at 2 weeks post‐MI. C) Quantitative analysis of cardiac systolic function parameters including ejection fraction (EF, t = 3.107) and fractional shortening (FS, t = 3.100) in ADAMTS1 CKO ( n = 9) and ADAMTS1 fl/fl mice ( n = 7). D) Echocardiographic measurements of (a) end‐diastolic diameter (EDD, t = 3.689) and end‐systolic diameter (ESD, t = 3.893), and (b) end‐diastolic volume (EDV, t = 3.476) and end‐systolic volume (ESV, t = 3.803) in ADAMTS1 CKO and ADAMTS1 fl/fl mice. E) Representative B‐mode echocardiographic images showing ventricular wall expansion and regional myocardial motion in ADAMTS1 CKO and ADAMTS1 fl/fl mice. F) Transmission electron microscopy (TEM) images of scar tissue and quantitative analysis of fibril diameter and Young's modulus ADAMTS1 CKO and ADAMTS1 fl/fl mice ( n = 3 per group; fibril diameter: t = 14.71; Young's modulus: t = 8.337). Scale bars = 500 nm. G) Representative Masson's trichrome staining of cardiac cross‐sections from apex to base showing fibrotic areas (blue) in ADAMTS1 CKO and ADAMTS1 fl/fl mice. Scale bars = 1 mm. H) Quantitative analysis of fibrosis severity classified as mild, moderate, and severe, with dot plot showing distribution of fibrotic area percentage in ADAMTS1 CKO and ADAMTS1 fl/fl mice ( n = 6 per group; t = 2.314). Statistical analyses were performed using unpaired t ‐test. Data are presented as mean ± SD.

Article Snippet: To investigate ADAMTS1‐mediated ITGα8 activation, two experiments were conducted: (1) NRCFs were treated with recombinant human ADAMTS1 (100 ng mL −1 , R&D Systems) or PBS control, followed by co‐immunoprecipitation and IF staining to assess ADAMTS1‐ITGα8 interaction and activation. (2) NRCFs were cultured on 1%, 2%, or 3% hydrogels for 24 h to evaluate ITGα8 activation in response to altered substrate stiffness using IF staining.

Techniques: Knock-Out, Transmission Assay, Electron Microscopy, Staining

Journal: Advanced Science

Article Title: Adamts1 Exacerbates Post‐Myocardial Infarction Scar Formation via Mechanosensing of Integrin α8

doi: 10.1002/advs.202504138

Figure Lengend Snippet: Mechanical activation of ITGα8 by Adamts1 regulates scar formation. A) Schematic illustration of experimental workflow for combined transcriptome and proteome analysis, showing tissue isolation, processing, and multi‐omics analysis pipeline. B) Venn diagram showing overlap between transcriptome (809 genes) and proteome (113 proteins) datasets, and heatmap displaying opposing expression patterns of selected genes in ADAMTS1 Tie versus ADAMTS1 CKO mice ( n = 5 and 3). C) Transcript level of ITGa8 at different time points post‐MI ( n = 6 per group). 3 days: F (2,15) = 40.74, p = 8.66e‐007, 7 days: F (2, 15) = 221.1, p = 7.41e‐012, 14 days: F (2, 15) = 186.3, p = 2.56e‐011. 28 days: F (2, 15) = 138.0, p = 2.19e‐010. D) Western blot analysis of ITGα8 and Adamts1 protein expression in ADAMTS1 Tie and ADAMTS1 CKO mice ( n = 6); t (Adamts1 Tie ) = 4.562, t (ADAMTS1 CKO ) = 9.329. E) Immunofluorescence (IF) staining of ITGα8 in cardiac fibroblasts treated with recombinant human Adamts1 protein ( n = 5, at least six fields per replicate, t = 0.7064). F) Co‐immunoprecipitation analysis examining protein interactions between Adamts1 and ITGα8. G) Schematic diagram of tunable stiffness sodium alginate (SA) hydrogel preparation and mechanical characterization. H) IF staining of cardiac fibroblasts cultured on 1% and 3% SA hydrogels. Statistical analysis was performed using unpaired t tests (C and D). Data are presented as the mean ± SD. Adamts1, a disintegrin and metalloproteinase with thrombospondin motif 1; VEC, vector; CKO, conditional knockout; Tnn, tenascin n; Snca, synuclein α; Slc4a1, solute carrier family 4 member 1; Slc43a1, solute carrier family 43 member 1; Mfap4, microfibril associated protein 4; Lum, lumican; ITGα8, integrin subunit α8; Dpt, dermatopontin; rhAdamts1, recombinant human Adamts1 protein.

Article Snippet: To investigate ADAMTS1‐mediated ITGα8 activation, two experiments were conducted: (1) NRCFs were treated with recombinant human ADAMTS1 (100 ng mL −1 , R&D Systems) or PBS control, followed by co‐immunoprecipitation and IF staining to assess ADAMTS1‐ITGα8 interaction and activation. (2) NRCFs were cultured on 1%, 2%, or 3% hydrogels for 24 h to evaluate ITGα8 activation in response to altered substrate stiffness using IF staining.

Techniques: Activation Assay, Isolation, Biomarker Discovery, Expressing, Western Blot, Immunofluorescence, Staining, Recombinant, Immunoprecipitation, Cell Culture, Plasmid Preparation, Knock-Out

Journal: Advanced Science

Article Title: Adamts1 Exacerbates Post‐Myocardial Infarction Scar Formation via Mechanosensing of Integrin α8

doi: 10.1002/advs.202504138

Figure Lengend Snippet: ITGα8 deficiency rescues cardiac dysfunction and reduces infarct size in Adamts1 overexpression mice post‐myocardial infarction. A) Representative M‐mode and B‐mode long‐axis echocardiographic images showing cardiac function in MI+ADAMTS1 Tie mice with ITGα8 fl/fl and ITGα8 CKO backgrounds at 2 weeks post‐MI. B) Quantitative analysis of cardiac systolic function parameters including ejection fraction (EF, t = 6.768) and fractional shortening (FS, t = 6.175) in MI+Adamts1 Tie mice with ITGα8 fl/fl and ITGα8 CKO backgrounds ( n = 6 for ITGα8 fl/fl , n = 9 for ITGα8 CKO ). C) Echocardiographic measurements of end‐diastolic diameter (EDD, t = 5.313) and end‐systolic diameter (ESD, t = 8.304) in the two experimental groups. D) Quantitative analysis of end‐diastolic volume (EDV, t = 5.359) and end‐systolic volume (ESV, t = 7.977) in ITGα8 fl/fl and ITGα8 CKO mice with Adamts1 overexpression. E) Representative 3D speckle tracking echocardiographic images showing wall displacement patterns in ITGα8 fl/fl and ITGα8 CKO mice with Adamts1 overexpression. F) Quantitative analysis of longitudinal strain at end‐apex ( t = 5.623) and radial strain at end‐apex ( t = 5.629) in the two experimental groups. G) Representative Evans blue and 2,3,5‐triphenyltetrazolium chloride (TTC) staining of cardiac sections from ligation site to apex. Non‐ischemic myocardium appears blue, infarct area (IA) appears pale/white, and area at risk (AAR) appears red. Scale bar = 1 cm. H) Quantitative analysis of IA to area at risk ratio (IA/AAR) and area at risk to left ventricle ratio (AAR/LV) in ITGα8 fl/fl and ITGα8 CKO mice with Adamts1 overexpression ( n = 6 per group). Statistical analyses were performed using unpaired t ‐test. Data are presented as mean ± SD.

Article Snippet: To investigate ADAMTS1‐mediated ITGα8 activation, two experiments were conducted: (1) NRCFs were treated with recombinant human ADAMTS1 (100 ng mL −1 , R&D Systems) or PBS control, followed by co‐immunoprecipitation and IF staining to assess ADAMTS1‐ITGα8 interaction and activation. (2) NRCFs were cultured on 1%, 2%, or 3% hydrogels for 24 h to evaluate ITGα8 activation in response to altered substrate stiffness using IF staining.

Techniques: Over Expression, Staining, Ligation

Journal: Cancers

Article Title: Activation of G Protein-Coupled Estrogen Receptor Induces p53 and ADAMTS1 to Inhibit Tumor Growth and Suppress Liver Cancer Metastasis

doi: 10.3390/cancers17162623

Figure Lengend Snippet: ADAMTS1 and GPER agonists inhibit the invasive ability of SK-Hep-1 cells. ( a ) Genes with RNA expression increased by more than 5.0-fold ( p < 0.0005) and protein expression increased by more than 1.6-fold ( p < 0.001). ( b ) ADAMTS1-treated SK-Hep-1 cell lysates obtained after 48 h. The expression of EMT-related proteins was measured through Western blotting. The amount of each protein was calculated compared to β-actin using ImageJ. Data are presented as mean ± standard deviation ( n = 3). ( c ) An invasion assay was performed with ADAMTS1-treated SK-Hep-1 cells after 48 h. Photos were observed via microscopy (10×). Graph representing the area of the invading cell calculated using ImageJ. Data are presented as mean ± standard deviation ( n = 3). * p < 0.01 (two-tailed unpaired t -test). ( d ) The protein expression detected in SK-Hep-1 cells treated with G1 (1 µM) and tamoxifen (TA) (17 µM). Cell lysates from G1 and TA-treated SK-Hep-1 cells were collected after 48 h. ( e ) The invasion was confirmed with G1- and G15-treated SK-Hep-1 cells for 48 h. Photos were observed via microscopy (10×). The graph shows the area of invaded cells calculated using ImageJ. Data are presented as mean ± standard deviation ( n = 3) ** p < 0.01 (one-way ANOVA with Dunnett’s post hoc test). The original images of the Western Blotting figures can be found in .

Article Snippet: In ADAMTS1 and tamoxifen treatment, cells were treated with 10, 50, 100, 200, and 400 ng of recombinant human ADAMTS1 protein (R&D systems, Minneapolis, MN, USA) dissolved in DPBS and 17 μM of tamoxifen (Sigma-Aldrich) dissolved in 0.2% DMSO for 48 h. The control group was treated with 0.2% DMSO alone.

Techniques: RNA Expression, Expressing, Western Blot, Standard Deviation, Invasion Assay, Microscopy, Two Tailed Test

Journal: Cancers

Article Title: Activation of G Protein-Coupled Estrogen Receptor Induces p53 and ADAMTS1 to Inhibit Tumor Growth and Suppress Liver Cancer Metastasis

doi: 10.3390/cancers17162623

Figure Lengend Snippet: GPER agonist increases ADAMTS1 expression and inhibits metastasis. ( a ) The SK-Hep-1 cell-derived xenograft model and the control group received intraperitoneal administration of G1 at 10 mg/kg thrice weekly. Tumor size and liver metastases were compared between the G1 and control groups (arrow: metastatic lesion). Data are presented as mean ± standard deviation ( n = 5) * p < 0.05 (two-tailed unpaired t -test). ( b ) ADAMTS1, E-cadherin, Vimentin, and PCNA levels were measured in the transplanted subcutaneous tumor. The ratio of these protein levels to β-actin was calculated using ImageJ. Data are presented as mean ± standard deviation ( n = 6) * p < 0.05, ** p < 0.01 (two-tailed unpaired t -test). ( c ) Immunohistochemical staining for ADAMTS1 in the subcutaneously implanted tumor. The area of ADAMTS1 protein levels was calculated using ImageJ. Data are presented as mean ± standard deviation ( n = 3) * p < 0.05 (two-tailed unpaired t -test). ( d ) The overall survival rates of liver cancer patients were compared with GPER and ADAMTS1 expression levels in men and women. In the KM plotter database, the gene IDs of GPER and ADAMTS1 were 2852 and 9510, respectively. HR, hazard ratio. Data were analyzed using the optimal threshold as the cut-off. The original images of the Western Blotting figures can be found in .

Article Snippet: In ADAMTS1 and tamoxifen treatment, cells were treated with 10, 50, 100, 200, and 400 ng of recombinant human ADAMTS1 protein (R&D systems, Minneapolis, MN, USA) dissolved in DPBS and 17 μM of tamoxifen (Sigma-Aldrich) dissolved in 0.2% DMSO for 48 h. The control group was treated with 0.2% DMSO alone.

Techniques: Expressing, Derivative Assay, Control, Standard Deviation, Two Tailed Test, Immunohistochemical staining, Staining, Western Blot

Journal: Cancers

Article Title: Activation of G Protein-Coupled Estrogen Receptor Induces p53 and ADAMTS1 to Inhibit Tumor Growth and Suppress Liver Cancer Metastasis

doi: 10.3390/cancers17162623

Figure Lengend Snippet: ADAMTS1 and GPER agonists inhibit the invasive ability of SK-Hep-1 cells. ( a ) Genes with RNA expression increased by more than 5.0-fold ( p < 0.0005) and protein expression increased by more than 1.6-fold ( p < 0.001). ( b ) ADAMTS1-treated SK-Hep-1 cell lysates obtained after 48 h. The expression of EMT-related proteins was measured through Western blotting. The amount of each protein was calculated compared to β-actin using ImageJ. Data are presented as mean ± standard deviation ( n = 3). ( c ) An invasion assay was performed with ADAMTS1-treated SK-Hep-1 cells after 48 h. Photos were observed via microscopy (10×). Graph representing the area of the invading cell calculated using ImageJ. Data are presented as mean ± standard deviation ( n = 3). * p < 0.01 (two-tailed unpaired t -test). ( d ) The protein expression detected in SK-Hep-1 cells treated with G1 (1 µM) and tamoxifen (TA) (17 µM). Cell lysates from G1 and TA-treated SK-Hep-1 cells were collected after 48 h. ( e ) The invasion was confirmed with G1- and G15-treated SK-Hep-1 cells for 48 h. Photos were observed via microscopy (10×). The graph shows the area of invaded cells calculated using ImageJ. Data are presented as mean ± standard deviation ( n = 3) ** p < 0.01 (one-way ANOVA with Dunnett’s post hoc test). The original images of the Western Blotting figures can be found in .

Article Snippet: In ADAMTS1 and tamoxifen treatment, cells were treated with 10, 50, 100, 200, and 400 ng of recombinant human ADAMTS1 protein (R&D systems, Minneapolis, MN, USA) dissolved in DPBS and 17 μM of tamoxifen (Sigma-Aldrich) dissolved in 0.2% DMSO for 48 h. The control group was treated with 0.2% DMSO alone.

Techniques: RNA Expression, Expressing, Western Blot, Standard Deviation, Invasion Assay, Microscopy, Two Tailed Test

Journal: Cancers

Article Title: Activation of G Protein-Coupled Estrogen Receptor Induces p53 and ADAMTS1 to Inhibit Tumor Growth and Suppress Liver Cancer Metastasis

doi: 10.3390/cancers17162623

Figure Lengend Snippet: GPER agonist increases ADAMTS1 expression and inhibits metastasis. ( a ) The SK-Hep-1 cell-derived xenograft model and the control group received intraperitoneal administration of G1 at 10 mg/kg thrice weekly. Tumor size and liver metastases were compared between the G1 and control groups (arrow: metastatic lesion). Data are presented as mean ± standard deviation ( n = 5) * p < 0.05 (two-tailed unpaired t -test). ( b ) ADAMTS1, E-cadherin, Vimentin, and PCNA levels were measured in the transplanted subcutaneous tumor. The ratio of these protein levels to β-actin was calculated using ImageJ. Data are presented as mean ± standard deviation ( n = 6) * p < 0.05, ** p < 0.01 (two-tailed unpaired t -test). ( c ) Immunohistochemical staining for ADAMTS1 in the subcutaneously implanted tumor. The area of ADAMTS1 protein levels was calculated using ImageJ. Data are presented as mean ± standard deviation ( n = 3) * p < 0.05 (two-tailed unpaired t -test). ( d ) The overall survival rates of liver cancer patients were compared with GPER and ADAMTS1 expression levels in men and women. In the KM plotter database, the gene IDs of GPER and ADAMTS1 were 2852 and 9510, respectively. HR, hazard ratio. Data were analyzed using the optimal threshold as the cut-off. The original images of the Western Blotting figures can be found in .

Article Snippet: In ADAMTS1 and tamoxifen treatment, cells were treated with 10, 50, 100, 200, and 400 ng of recombinant human ADAMTS1 protein (R&D systems, Minneapolis, MN, USA) dissolved in DPBS and 17 μM of tamoxifen (Sigma-Aldrich) dissolved in 0.2% DMSO for 48 h. The control group was treated with 0.2% DMSO alone.

Techniques: Expressing, Derivative Assay, Control, Standard Deviation, Two Tailed Test, Immunohistochemical staining, Staining, Western Blot

Journal: Cell

Article Title: A mast cell receptor mediates post-stroke brain inflammation via a dural-brain axis

doi: 10.1016/j.cell.2025.06.045

Figure Lengend Snippet: (A) Outline of UBC-GFP skull transplant experiment (BioRender). (B-C) (B) Absolute GFP-positive neutrophil count and (C) percentage of dural neutrophils that are GFP-positive in WT/ Mrgprb2 −/− mice 48h post-tMCAO (WT n =6, Mrgprb2 −/− n =6). (D) Percentage of skull bone marrow neutrophils that are GFP-positive in WT/ Mrgprb2 −/− mice 48h post-tMCAO (WT n =5, Mrgprb2 −/− n =6). (E) Representative immunofluorescence image of WT recipient right brain hemisphere 48h post-tMCAO. left, Ly6G denotes neutrophils, middle , GFP denotes cells recruited from skull bone marrow, and right, Merged channels with DAPI. White arrows indicate cells with dual Ly6G/GFP stain. Scale bar=20 μm. (F) Absolute GFP-positive neutrophil count in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− brains (WT n =5, Mrgprb2 −/− n =6). (G) Percentage of contralateral/stroke brain hemispheres neutrophils that are GFP-positive in WT/ Mrgprb2 −/− brains (WT n =5, Mrgprb2 −/− n =6). (H) Semaphorin3a protein levels in WT/ Mrgprb2 −/− leptomeninges after sham/tMCAO (WT sham n =7, Mrgprb2 −/− sham n= 4, WT tMCAO n =10, Mrgprb2 −/− tMCAO n= 9). (I) Neutrophil count/frame in dura overlaying the contralateral/stroke brain hemisphere of vehicle/Sema3-I treated Mrgprb2 −/− mice 48h post-tMCAO. (vehicle n =6, Sema3a-I n =5). (J) Absolute neutrophil count in contralateral/stroke brain hemispheres of vehicle/Sema3a-I treated Mrgprb2 −/− mice 48h post-tMCAO (vehicle n =7, Sema3a-I n =7). (K) left, Representative T2-weighted MRIs and right, stroke volume of vehicle/Sema3a-I treated Mrgprb2 −/− mice 48h post-tMCAO (vehicle n =4, Sema3a-I n =5). (L) Vehicle/Sema3a-I treated Mrgprb2 −/− mice neurological scores pre- and post-tMCAO. Scores normalized to vehicle-treated mice within cohorts (vehicle n =6, Sema3a-I n =6). (M) Recombinant SEMA3A-Fc incubated with dermal fibroblast lysate, WT mast cell lysate, or ADAMTS1 as a positive control. Cleaved, inactive SEMA3A-Fc indicated by smaller 65kDa band. Statistical analyses: Mann-Whitney test (B-C), two-sided Student’s t-test (D, K), two-way ANOVA with Sidak’s multiple comparisons test (F,G,I-J, and L), and two-way ANOVA with Tukey’s multiple comparisons test (H). Statistical test for F, and L was performed on log-transformed data to adjust for non-normality. Bar graphs indicate mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: For positive control, 0.75μg of recombinant ADAMTS1 (R&D Systems) was placed with 0.75μg of semaphorin in 40μL of 50mM Tris pH 7.4, 10mM CaCl 2 , 80mM NaCl.

Techniques: Immunofluorescence, Staining, Recombinant, Incubation, Positive Control, MANN-WHITNEY, Transformation Assay

Journal: Cellular & molecular biology letters

Article Title: The oncogenic ADAMTS1-VCAN-EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma.

doi: 10.1186/s11658-024-00643-0

Figure Lengend Snippet: Fig. 5 The metalloproteinase activity of ADAMTS1 is pivotal in stimulating the secretion of cleaved versican, activating the epidermal growth factor receptor (EGFR), promoting invasion, and conferring anoikis resistance in renal cell carcinoma (RCC) cells. A A schematic representation depicting two variants of the recombinant ADAMTS1 protein: one containing the metalloproteinase domain (ZnMc) and the other lacking it (TSP), along with a mutant ADAMTS1 expression construct (E402Q). B–E Treatment of Caki-1 cells with or without the recombinant ZnMc or TSP protein (40 nM) for 24 or 48 h. Subsequently, secretion of cleaved versican, activation of EGFR signal cascades, invasive abilities, and anoikis were respectively assessed using dot blot (B), western blotting (C), Matrigel invasion (D), and CCK8 assays. F–I Cleaved versican secretion (F), EGFR activation (G), invasive abilities (H), and anoikis (I) were evaluated in Caki-1 and 786-O cells after transducing wild-type (WT) ADAMTS1, ADAMTS1/E402Q, or a control vector. In D, E, H, and I, values are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group; #p < 0.05, ###p < 0.001, compared with the WT ADAMTS1-overexpressing group

Article Snippet: Recombinant human ADAMTS1 (2197-AD) containing a zinc-dependent metalloprotease (ZnMc) domain was purchased from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Recombinant, Mutagenesis, Expressing, Construct, Activation Assay, Dot Blot, Western Blot, Control, Plasmid Preparation

Journal: Cellular & molecular biology letters

Article Title: The oncogenic ADAMTS1-VCAN-EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma.

doi: 10.1186/s11658-024-00643-0

Figure Lengend Snippet: Fig. 1 Elevated ADAMTS1 expression in renal cell carcinoma (RCC) tissues is associated with a poor prognosis. A ADAMTS1 mRNA levels in RCC tissues were significantly higher than those in normal renal tissues (p = 0.033) as determined by TNMplot (https://tnmplot.com/analysis/). B ADAMTS1 expression was assessed in 72 matched clear cell (cc)RCC tissues and their corresponding normal tissues using data from the GSE53757 ccRCC expression microarray dataset. C The left panel of the Uniform Manifold Approximation and Projection (UMAP) plot illustrates the distribution of tumor cells and benign epithelial cells based on single-cell RNA sequencing (scRNA-seq) profiles. The UMAP plot in the middle panel shows the expression distribution of ADAMTS1. The violin plot in right panel indicates that ADAMTS1 exhibits a significant elevation in tumor cells compared with benign epithelial cells, with a fold change (FC) of 1.27 and a false discovery rate (FDR) of less than 0.01. D Kaplan–Meier analysis depicts overall survival (OS) rates in patients with ccRCC based on high or low ADAMTS1 expression using data from TCGA

Article Snippet: Recombinant human ADAMTS1 (2197-AD) containing a zinc-dependent metalloprotease (ZnMc) domain was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Microarray, RNA Sequencing

Journal: Cellular & molecular biology letters

Article Title: The oncogenic ADAMTS1-VCAN-EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma.

doi: 10.1186/s11658-024-00643-0

Figure Lengend Snippet: Fig. 2 ADAMTS1 expression promotes anoikis resistance of renal cell carcinoma (RCC) via inhibiting Bid, Bim, and Bak. A A western blot analysis was conducted to assess ADAMTS1 expression in Caki-1 and 786-O cells following transduction with either ADAMTS1 short hairpin (sh)RNA (left) or an ADAMTS1-expressing vector (right). B Cell viability of suspended Caki-1 and 786-O cells was evaluated using a CCK8 assay at 24 and 48 h post-stable overexpression (right) or knockdown (left) of ADAMTS1. Data are presented as the mean ± SD of three independent experiments. * p < 0.05, compared with the control group. C Dot plot demonstrated the correlation between the single sample gene set enrichment analysis (ssGSEA) score of “negative regulation of anoikis” and ADAMTS1 expression in TCGA-KIRC patients. A Pearson correlation was performed to evaluate their association and significance. D Western blot analysis of intrinsic apoptosis-related proteins (Bad, Bcl-2, Bak, Bid, Bim, and PARP) in suspended Caki-1 cells manipulated with ADAMTS1. GAPDH served as a loading control. E and F Dissemination of RCC cells in zebrafish embryos. Caki-1 cells with ADAMTS1-knockdown were implanted into zebrafish embryos at 48 h post fertilization. Tumor cell dissemination was observed at 2 days post injection (dpi), with disseminated tumor foci indicated by white arrowheads on the trunk and end-tail (E). Integrated densities of Caki-1 metastatic tumor cells in the zebrafish trunk and end-tail at 2 dpi were quantified, with the mean value of the integrated density in the shCtl group set to onefold (F)

Article Snippet: Recombinant human ADAMTS1 (2197-AD) containing a zinc-dependent metalloprotease (ZnMc) domain was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Transduction, Plasmid Preparation, CCK-8 Assay, Over Expression, Knockdown, Control, Injection

Journal: Cellular & molecular biology letters

Article Title: The oncogenic ADAMTS1-VCAN-EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma.

doi: 10.1186/s11658-024-00643-0

Figure Lengend Snippet: Fig. 5 The metalloproteinase activity of ADAMTS1 is pivotal in stimulating the secretion of cleaved versican, activating the epidermal growth factor receptor (EGFR), promoting invasion, and conferring anoikis resistance in renal cell carcinoma (RCC) cells. A A schematic representation depicting two variants of the recombinant ADAMTS1 protein: one containing the metalloproteinase domain (ZnMc) and the other lacking it (TSP), along with a mutant ADAMTS1 expression construct (E402Q). B–E Treatment of Caki-1 cells with or without the recombinant ZnMc or TSP protein (40 nM) for 24 or 48 h. Subsequently, secretion of cleaved versican, activation of EGFR signal cascades, invasive abilities, and anoikis were respectively assessed using dot blot (B), western blotting (C), Matrigel invasion (D), and CCK8 assays. F–I Cleaved versican secretion (F), EGFR activation (G), invasive abilities (H), and anoikis (I) were evaluated in Caki-1 and 786-O cells after transducing wild-type (WT) ADAMTS1, ADAMTS1/E402Q, or a control vector. In D, E, H, and I, values are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group; #p < 0.05, ###p < 0.001, compared with the WT ADAMTS1-overexpressing group

Article Snippet: Recombinant human ADAMTS1 (2197-AD) containing a zinc-dependent metalloprotease (ZnMc) domain was purchased from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Recombinant, Mutagenesis, Expressing, Construct, Activation Assay, Dot Blot, Western Blot, Control, Plasmid Preparation

Journal: Cellular & molecular biology letters

Article Title: The oncogenic ADAMTS1-VCAN-EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma.

doi: 10.1186/s11658-024-00643-0

Figure Lengend Snippet: Fig. 6 Tissue inhibitor of metalloproteinase 3 (TIMP3) expression reverses ADAMTS1-induced versican cleavage, epidermal growth factor receptor (EGFR) activation, and invasion and anoikis resistance of renal cell carcinoma (RCC) cells. A The pcDNA3-TIMP3 plasmid was transfected into ADAMTS1-overexpressing Caki-1 and 786-O cells as indicated, followed by measurement of TIMP3 mRNA levels using an RT–qPCR. Quantitative results of TIMP3 mRNA levels were normalized to GAPDH mRNA levels. B–E Evaluation of cleaved versican secretion B, EGFR activation C, invasive abilities D, and anoikis (E in RCC cells overexpressing ADAMTS1, subsequent to transfection with either a vector control or TIMP3 plasmid. In D and E, the values are presented as the mean ± SD of three independent experiments. *p < 0.05, ***p < 0.001, compared with the vector control group; #p < 0.05, ###p < 0.001, compared with the ADAMTS1-overexpressing group. F Comparative analysis of TIMP3 mRNA levels among normal renal tissues, primary RCC, and metastatic RCC as determined by TNMplot. G Examination of TIMP3 gene expression levels in RCC from TCGA based on the pathological tumor (T), node (N), and metastasis (M) stages

Article Snippet: Recombinant human ADAMTS1 (2197-AD) containing a zinc-dependent metalloprotease (ZnMc) domain was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activation Assay, Plasmid Preparation, Transfection, Quantitative RT-PCR, Control, Gene Expression

Journal: Cellular & molecular biology letters

Article Title: The oncogenic ADAMTS1-VCAN-EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma.

doi: 10.1186/s11658-024-00643-0

Figure Lengend Snippet: Fig. 7 ADAMTS1 is associated with p53 to modulate epidermal growth factor receptor (EGFR) promoter activity and expression in renal cell carcinoma (RCC) cells. A Luciferase reporters were transfected into Caki-1 and 786-O cells with or without ADAMTS1 manipulation. All luciferase activity was normalized to Renilla reporter activity. The normalized reporter activity in cells transfected with a control vector was considered as onefold, and the relative fold change for each construct (ADAMTS1-flag or shADAMTS1) was further normalized to that of the control. B Reporters were transfected into Caki-1 and 786-O cells and treated with conditioned media (CM) collected from ADAMTS1-overexpressing or control Caki-1 and 786-O cells. The normalized reporter activity in cells treated with CM collected from control cells was considered onefold, and the relative fold change of RCC-derived CM treatment was calculated. The values are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group. C Western blotting analysis of ADAMTS1 expression levels in cytoplasmic and nuclear fractions from Caki-1 and 786-O cells transfected with ADAMTS1-flag or a control vector. D The immunocomplex was precipitated from ADAMTS1-overexpressing Caki-1 cell lysates with an ADAMTS1 antibody and analyzed by western blotting to detect the associations of ADAMTS1 with indicated transcription factors (p53, WT1, and c-Jun). A normal IgG antibody was used as an immunoprecipitation (IP) control, and 10% whole-cell lysate was used as the input. E Western blot analysis of p53, ADAMTS1, p-EGFR, and EGFR levels in Caki-1 and 786-O cells after transducing ADAMTS1-flag or a control vector and treatment with the p53 inhibitor, pifithrin (PFT)-α (10 µM), or the vehicle for 24 h

Article Snippet: Recombinant human ADAMTS1 (2197-AD) containing a zinc-dependent metalloprotease (ZnMc) domain was purchased from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Expressing, Luciferase, Transfection, Control, Plasmid Preparation, Construct, Derivative Assay, Western Blot, Immunoprecipitation

Journal: Cellular & molecular biology letters

Article Title: The oncogenic ADAMTS1-VCAN-EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma.

doi: 10.1186/s11658-024-00643-0

Figure Lengend Snippet: Fig. 8 Schematic presentation depicting the ADAMTS1–VCAN–EGFR axis in promoting the anoikis resistance and invasive abilities of renal cell carcinoma (RCC). ADAMTS1 mediates the proteolytic cleavage of VCAN V1, thereby activating EGFR cascades to promote anoikis resistance and invasion of RCC cells. Moreover, ADAMTS1 interacts with p53 to modulate EGFR expression. EGFR-driven signaling might potentially reinforce ADAMTS1 expression through positive feedback. Bold dashed oval and arrow indicate hypothetical molecules and pathways that participate in ADAMTS1-mediated EGFR activation or expression. The cyclic elevation of ADAMTS1 and EGFR exacerbates anoikis resistance and invasiveness in RCC cells

Article Snippet: Recombinant human ADAMTS1 (2197-AD) containing a zinc-dependent metalloprotease (ZnMc) domain was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activation Assay