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: ADAMTS1 levels in clinical serum samples were measured using an ADAMTS1 ELISA kit (CSB-E15060h, CUSABIO) following manufacturer’s protocol.

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: ADAMTS1 levels in clinical serum samples were measured using an ADAMTS1 ELISA kit (CSB-E15060h, CUSABIO) following manufacturer’s protocol.

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: ADAMTS1 levels in clinical serum samples were measured using an ADAMTS1 ELISA kit (CSB-E15060h, CUSABIO) following manufacturer’s protocol.

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: ADAMTS1 levels in clinical serum samples were measured using an ADAMTS1 ELISA kit (CSB-E15060h, CUSABIO) following manufacturer’s protocol.

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: ADAMTS1 levels in clinical serum samples were measured using an ADAMTS1 ELISA kit (CSB-E15060h, CUSABIO) following manufacturer’s protocol.

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: ADAMTS1 levels in clinical serum samples were measured using an ADAMTS1 ELISA kit (CSB-E15060h, CUSABIO) following manufacturer’s protocol.

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: ADAMTS1 levels in clinical serum samples were measured using an ADAMTS1 ELISA kit (CSB-E15060h, CUSABIO) following manufacturer’s protocol.

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: ADAMTS1 levels in clinical serum samples were measured using an ADAMTS1 ELISA kit (CSB-E15060h, CUSABIO) following manufacturer’s protocol.

Techniques: Ubiquitin Proteomics

Journal: Cell death & disease

Article Title: Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

doi: 10.1038/s41419-024-06452-9

Figure Lengend Snippet: Fig. 1 ADAMTS1 expression promotes invasion of oral squamous cell carcinoma (SCC; OSCC) cells and correlates with poor survival outcomes in patients with head and neck SCC (HNSCC). A ADAMTS1 transcripts in normal and HNSCC tissues were analyzed using data from TCGA. Statistical significance was analyzed by a Wilcoxon signed-rank test. B Kaplan–Meier analysis of overall survival (OS) and disease-specific survival (DSS) rates in patients with HNSCC presenting with high or low ADAMTS1 expression using data from TCGA. C Endogenous protein levels of ADAMTS1 in OSCC cell lines (HSC-3, HSC-3M, SCC9, and SAS) were detected by a Western blot analysis. D In vitro invasive abilities of OSCC cell lines were analyzed by a Matrigel-invasion assay. E Western blot analysis of ADAMTS1 expressions in HSC-3 (left) and HSC-3M (right) cells respectively expressing ADAMTS1-flag and ADAMTS1 shRNAs. F Invasive abilities of HSC-3 and HSC-3M cells were determined by a Matrigel-invasion assay after infecting cells with a lentivirus carrying ADAMTS1-flag, ADAMTS1 shRNAs, ADAMTS1 shRNA co-expressing ADAMTS1-flag, or their respective control vectors. Upper panel: representative photomicrographs (200×). Lower panel: quantitative values from counting of invaded cells presented as the mean ± SD of three independent experiments. ***p < 0.001, compared to the respective control groups. ##p < 0.01, compared to ADAMTS1 shRNA-infected only group. G, H Proliferation rates and colony-formation capacities of ADAMTS1-manipulated HSC-3 and HSC-3M cells were respectively determined using MTS and colony-forming assays. ADAMTS1 expression had no obvious effect on cell proliferation rates during 24–96 h (G) or the colony-formation capacity during 7‒10 days (H). ns not significant.

Article Snippet: An antibody specific for ADAMTS1 (AF5867) was purchased from R&D Systems, while those for L1CAM (sc514360) and TGF-β (sc130348) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, In Vitro, Invasion Assay, shRNA, Control, Infection

Journal: Cell death & disease

Article Title: Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

doi: 10.1038/s41419-024-06452-9

Figure Lengend Snippet: Fig. 2 ADAMTS1 expression accelerates tumor growth and cervical lymph node (LN) metastasis of oral squamous cell carcinoma (OSCC) in orthotopic mouse models. Male NOD/SCID mice were orthotopically injected with luciferase-tagged and ADAMTS1-overexpressing HSC-3 cells or ADAMTS1-knockdown HSC-3M cells. A, C Whole-body bioluminescence imaging was conducted each week for 5 weeks after injecting ADAMTS1-manipulated cells into mice. B, D Quantitative analysis of Xenogen imaging signal intensity (photons/s/cm2/sr) every week. *p < 0.05, ***p < 0.001, compared to the control group. Occurrence of cervical LN metastasis in NOD/SCID mice implanted with HSC-3/ADAMTS1 (E), HSC-3M/shADAMTS1 (F), or their respective control cells. The appearance, number and volume of cervical LNs were photographed, enumerated, and measured after removal. Data are presented as the mean ± SD. ***p < 0.001 compared to the control group.

Article Snippet: An antibody specific for ADAMTS1 (AF5867) was purchased from R&D Systems, while those for L1CAM (sc514360) and TGF-β (sc130348) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Injection, Luciferase, Knockdown, Imaging, Control

Journal: Cell death & disease

Article Title: Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

doi: 10.1038/s41419-024-06452-9

Figure Lengend Snippet: Fig. 3 L1CAM is critical in ADAMTS1-modulated invasive ability of oral squamous cell carcinoma (SCC; OSCC) cells and was correlated with poor prognoses in patients with head and neck SCC (HNSCC). A, B HSC-3 or HSC-3M cells expressed ADAMTS1-flag, shADAMTS1, or their respective control as indicated. Cell lysates and conditioned media were collected to detect endogenous ADAMTS1 or L1CAM protein levels (A) and secreted soluble L1CAM (B), respectively using western blotting and Dot-blotting assays. C A L1CAM shRNA was transfected into ADAMTS1-overexpressing HSC-3 cells as indicated and subjected to Matrigel-invasion assays. D HSC-3 cells were treated with indicated concentrations of rhL1CAM for 24 h and subjected to Matrigel-invasion assays. C, D Multiples of differences are presented as the mean ± SD of three independent experiments. ***p < 0.001, compared to the control group; ###p < 0.001, compared to the ADAMTS1-overexpressing only group. E L1CAM expression was analyzed in 43 matched HNSCC tissues and their corresponding normal tissues using data from TCGA. F Kaplan–Meier analysis of overall survival (OS) and disease-specific survival (DSS) rates in patients with HNSCC presenting with high or low L1CAM expression using data from TCGA. G Correlation analysis of TCGA HNSCC databases (TCGA, PanCancer Atlas) using cBioPortal which revealed a positive correlation between expressions of ADAMTS1 and L1CAM. H Survival heat map showing the prognostic impacts of ADAMTS1 and L1CAM on 33 different cancer types according to the GEPIA2 database. HR hazard ratio. I Combined high ADAMTS1 expression and high L1CAM expression were correlated with the worst OS and DSS in patients with HNSCC compared to patients with other expression statuses of ADAMTS1 and L1CAM.

Article Snippet: An antibody specific for ADAMTS1 (AF5867) was purchased from R&D Systems, while those for L1CAM (sc514360) and TGF-β (sc130348) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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

Journal: Cell death & disease

Article Title: Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

doi: 10.1038/s41419-024-06452-9

Figure Lengend Snippet: Fig. 6 Targeting ADAMTS1 by apigenin (API) resulted in suppression of the invasion of oral squamous cell carcinoma (OSCC) cells. A HSC-3, HSC-3M, and SAS cells were treated with API at 40 μM for different durations, and ADAMTS1, L1CAM, and EGFR expressions were evaluated by Western blotting. B HSC-3M cells were treated with API or infected with ADAMTS1 shRNAs for 24 h, and cell-invasive abilities were measured by a Matrigel-invasion assay. C, D HSC-3 cells were transiently transfected with a vector control or ADAMTS1-flag followed by API or vehicle treatment for an additional 24 h. ADAMTS1 expression and invasive ability in cells were respectively detected by Western blotting (C) and Matrigel-invasion assays (D). B, D Representative photographs of invaded cells (left panel) and quantification of those cells (right panel). Data are presented as the mean ± SD of three independent experiments, *p < 0.05, ***p < 0.001 vs. control cells and ##p < 0.01, vs. ADAMTS1-overexpressing only cells.

Article Snippet: An antibody specific for ADAMTS1 (AF5867) was purchased from R&D Systems, while those for L1CAM (sc514360) and TGF-β (sc130348) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Infection, Invasion Assay, Transfection, Plasmid Preparation, Control, Expressing

Journal: Cell death & disease

Article Title: Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

doi: 10.1038/s41419-024-06452-9

Figure Lengend Snippet: Fig. 8 Schematic presentation depicting the ADAMTS1-L1CAM-EGFR axis in promoting the epithelial-mesenchymal transition (EMT) and metastasis of oral squamous cell carcinoma (OSCC). EGFR activation might be triggered by formation of the ADAMTS1-L1CAM-EGFR complex or through ADAMTS1-mediated TGF-β upregulation to subsequently induce L1CAM upregulation and L1CAM-integrin binding, resulting in induction of IL-1β secretion. Bold dashed ovals indicate hypothetical molecules that participate in the ADAMTS1-L1CAM axis to transactivate EGFR signaling, and EGFR-activated signaling may exert positive feedback regulation on ADAMTS1 expression. Cyclic increases in ADAMTS1 and EGFR activation lead to exacerbation of the EMT and invasive abilities of OSCC cells.

Article Snippet: An antibody specific for ADAMTS1 (AF5867) was purchased from R&D Systems, while those for L1CAM (sc514360) and TGF-β (sc130348) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: Relative gene expression of serglycin and ADAMTS1.

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Gene Expression

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: (A) ADAMTS1 mRNA levels in indicated NAF derivatives were analyzed and shown gradually increased in NAF 200N.E4 from P0 to P3 and maintained from P3 to P5. (B) MeDIP indicates that ADAMTS1 promoter-associated DNA methylation level was similar in NAF 200N.P10, CAF 199C.P10 and NAF 200N.E4.P3 cells. (C) Bisulfite sequencing indicates that ADAMTS1 promoter in CAF 199C.P10, NAF 200N.P10 and NAF 200N.E4.P3 cells was hypomethylated. Closed circle: methylated cytosine. (D) ADAMTS1 promoter-associated H3K27me3 and EZH2 binding was decreased in CAF 199C.P10 and NAF 200N.E4.P3 cells. (B and D) MeDIP (B) or ChIP assays (D) using antibody against H3ace, H3K4me3, H3K9me3, H3K27me3, H3K36me3, H3K79me3 or EZH2 was performed in NAF 200N.P10 (white bars), CAF 199C.P10 (black bars) and NAF 200N.E4.P3 (gray bars) cells, followed by real-time PCR analysis. (E) ADAMTS1 promoter-associated H3K27me3 and EZH2 binding were decreased in NAF 200N.E4.P3 and maintained through passages to NAF 200N.E4.P5. ChIP assays using antibody against H3K27me3 (black bars) or EZH2 (white bars) were performed in cells indicated, followed by real-time PCR analysis. (B, D and E) PCR amplification was performed with DNA primers against the ADAMTS1 promoter region from −431 bp to −217 bp. Data are shown as mean ± SD from triplicate experiments. Statistical significance was evaluated by Student's t-test. * P <0.05. NS: No significant difference.

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation Sequencing, Methylation, Binding Assay, Real-time Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: (A) Sequestration of the ADAMTS1 activity in the conditional medium derived from CAF 199C.P10 using an anti-ADAMTS1 antibody reduced invasion ability of MDA-MB-468 cells (left) and MDA-MB-231 cells (right) in an anti-ADAMTS1 Ab dose-dependent manner. (B) Neutralization of the ADAMTS1 activity in the conditional medium derived from NAF 200N.E4.P3 using an anti-ADAMTS1 antibody also reduced invasion ability of MDA-MB-468 cells (left) and MDA-MB-231 cells (right). (C) Western shows the efficient inhibition of ADAMTS1 protein level by three independent shRNAs. (D) The conditional medium derived from CAF 199C.P10 depleted of ADAMTS1 by shRNAs exhibited significantly lower activity for invasion of MDA-MB-468 cells (left) and MDA.MB-231 cells (right). (E) The conditional media derived from NAF 200N.P10 with exogenous ADAMTS1 expression (left, western) enhanced invasion of MDA-MB-468 cells (middle) and MDA-MB-231 cells (right). Data are shown as mean ± SD from triplicate experiments. Statistical significance was evaluated by Student's t-test. * P <0.05.

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Activity Assay, Derivative Assay, Neutralization, Western Blot, Inhibition, Expressing

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: (A) A scatter dot plot of ADAMTS1 mRNA levels in CAFs assessed using quantitative real-time RT-PCR analysis. The ADAMTS1 mRNA levels in CAFs of patients with lymph node metastasis (n = 27) were significantly higher than in those of patients without lymph node metastasis (n = 22) (P = 0.0003). Data are shown as mean ± SD of triplicate samples. Statistical significance was evaluated by Student's t-test. *** P<0.001. (B) The relationship between ADAMTS1 expression and lymph node metastasis was analyzed using Fisher's exact test. ADAMTS1 expression levels in CAFs derived from patients with ≥60% lymph node metastasis were significantly higher than those of patients with ≤60% lymph node metastasis or with zero lymph node metastasis ( P = 0.001). Relative quantity (RQ) of ADAMTS1 expression compared to beta-actin expression: +, 0.001≤RQ≤0.025; ++, 0.025

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Breast Cancer Cells Induce Stromal Fibroblasts to Secrete ADAMTS1 for Cancer Invasion through an Epigenetic Change

doi: 10.1371/journal.pone.0035128

Figure Lengend Snippet: (A–C) Quantitative real-time RT-PCR analysis revealed that ADAMTS1 mRNA levels were higher in CAFs than in the corresponding NAFs in ten breast cancer patients (A), and that ADAMTS1 mRNA levels in CAF 199C.P10 and NAF 200N.E4.P3 were higher than in NAF 200N.P10 and 200N.E1-E3.P3 (B). Similar results are shown using the pairs of CAF 244C/NAF 245N and CAF 259C/NAF 260N (C). (D) The ADAMTS1 protein level in NAF 200N.E4.P3, but not NAF 200N.E1-E3.P3, was enhanced to the similar level in CAF 199C.P10, compared to NAF 200N.P10. (E) ELISA indicated that the ADAMTS1 protein level in cultured medium derived from NAF 200N.E4.P3 was higher than NAF 200N.E1-E3.P3 and NAF 200N.P10. (F) ADAMTS1 mRNA level in SK-BR-3 cell-precocultured NAF 200N.S4.P3 was also higher than in NAF 200N.P10 and 200N.S1-S3.P3. Data are shown as mean ± SD from triplicate experiments. Statistical significance was evaluated by Student's t-test. * P <0.05.

Article Snippet: For the purpose of neutralizing ADAMTS1 activity, 0.3–7.5 μg of mouse monoclonal anti-human ADAMTS1 antibody (R&D Systems) or mouse IgG 2B (R&D Systems) was added to 100 μl of 100% confluent fibroblast-cultured medium.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay