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: Food Science & Nutrition

Article Title: Dihydroquercetin Attenuates Silica‐Induced Pulmonary Fibrosis by Modulating the Gut Microbiota and the Serum Metabolites in Mice

doi: 10.1002/fsn3.71389

Figure Lengend Snippet: DHQ treatment attenuated silica‐induced pulmonary fibrosis in C57/BL6 mice. (A, B) DHQ treatment increased the body weight and decreased pulmonary index in silicosis model mice. The changes of body weight (C) The levels of pro‐inflammatory cytokines (IL‐1β, TNF‐α, and TGF‐β) in serum from different groups at day 21 were detected by ELISA assay. (D) Representative pictures (×200) of HE‐stained and Masson‐stained lung sections from mice on day 21 were shown. Bar = 100 μm. (E, F) The inflammation and fibrosis score numbers of 0–3, corresponding to the grades of –, +, ++, and +++, were evaluated by experienced pathologists in a blinded fashion. (G) Representative results of western blot for α‐SMA, collagen I and fibronectin in lung tissues and the quantification of results. Data are shown as mean ± SD. All experiments were repeated three times. # p < 0.05, ## p < 0.01 vs. the control group; * p < 0.05, ** p < 0.01 vs. the SiO 2 group.

Article Snippet: Sigma‐Aldrich provided the SiO 2 (Cat#S5631) particles (around 80% diameter 1‐5 μm), which were filtered through sedimentation following Stokes' law, underwent acidic hydrolysis, and were baked overnight at 200°C for 16 h. Interleukin‐1β (IL‐1β) (CSB‐E08054m), tumor necrosis factor‐α (TNF‐α) (CSB‐E04741m), and transforming growth factor‐β (TGF‐β) (CSB‐E04726m) were acquired from Cusabio Biotechnology in Wuhan, China.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Control

Journal: International heart journal

Article Title: Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFβ-1.

doi: 10.1536/ihj.16-572

Figure Lengend Snippet: Figure 2. Reduction of TGFβ1 expression by downregulation of RyR2in cardiomyocytes under mechani- cal stretch. A: Measurement of RyR2 mRNA level in cultured cardiomyocytes by Real-Time PCR. B: Expres- sion of RyR2 protein level in cardiomyocytes with Western Blotting. C: TGFβ1 in supernates of cultured cardiomyocytes was detected with ELISA analysis. D: Expression of TGFβ1 protein level in cardiomyocytes with Western Blotting. E: Measurement of tgfb1 mRNA level in cultured cardiomyocytes using Real-Time PCR. Values are expressed as mean ± SEM obtained from 3 independent experiments. shRyR2 indicates RyR2 knockdown lentiviral particle-infected cardiomyocytes; MS, cardiomyocytes under mechanical stretch; MS + ryr2 KD, RyR2 knockdown cardiomyocytes under mechanical stretch. *P < 0.05 versus control.

Article Snippet: ELISA assay: Supernates of cultured cardiomyocytes were collected and examined by Rat Transforming Growth Factor β1 ELISA Kit (CUSABIO BIOTECH, China) following the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Infection, Control

Journal:

Article Title: NHERF2 Specifically Interacts with LPA 2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-?3 Activation

doi: 10.1128/MCB.24.11.5069-5079.2004

Figure Lengend Snippet: NHERF2 couples LPA2 to PLC-β3 specifically. (A) LPA2 forms a molecular complex with PLC-β3 in a NHERF2-dependent manner. The NHERF2 constructs (NHERF2, NHERF2 WT; NHERF2 ΔPDZ2, the second PDZ domain-deleted form) were cotransfected in combination with either Flag-PLC-β3 (left) or Flag-PLC-β1 (right) as indicated. After 2 days, COS-7 cells were washed and incubated with PBS containing 0.5 mM DSP (Pierce), which is a cell-permeable cross-linker, for 30 min. After the residual DSP was blocked with PBS containing 50 mM Tris buffer, the cleared lysates were subjected to a pull-down assay using GST-LPA2-CT immobilized onto GSH beads. After a washing step, the resulting precipitates were subjected to SDS-PAGE and then analyzed by Western blot analysis with anti-Flag antibody (top) and anti-NHERF2 antibody (middle) or by Ponceau S staining (bottom) as indicated. The results are representative of three independent experiments. (B) Specific coimmunoprecipitation of PLC-β3 and not of PLC-β1 with Flag LPA2. HeLa cells (at a density of 6 × 106 cells/150-mm dish) were infected with either the recombinant Flag-LPA2 adenovirus or control empty virus at an MOI of 10. At 24 h postinfection, the infected cells were incubated with PBS containing 0.5 mM DSP for 30 min. After the cell lysates were blocked with 50 mM Tris buffer, they were immunoprecipitated with anti-Flag antibody. After a washing step, the resulting precipitates were analyzed with the specific antibodies as indicated. (C) Gene silencing of PLC-β isoforms with specific siRNAs. HeLa cells were transfected with siRNAs directed against PLC-β1 or PLC-β3, along with control siRNA (Luciferase), as described in Materials and Methods. After 3 days, the HeLa cells were lysed and then analyzed by Western blot analysis with specific antibodies, as indicated. (D) PLC-β3 is functionally coupled to LPA2. At 24 h after siRNA transfection, the cells were split at a density of 2 × 105 cells/35-mm well and infected with recombinant adenovirus expressing Flag-LPA2 for 4 h. At 12 h after viral infection, the infected cells were labeled with 1 μCi of [3H]inositol/ml for 12 h and treated with either 0.1% BSA only (−) or 1 μM LPA-BSA conjugates (+). The generation of [3H]IPs was analyzed as described in Materials and Methods. The results are presented as means ± the SE of three experiments performed in duplicate.

Article Snippet: In addition, the specific antibodies to PLC-β1, PLC-β3, and phosphorylated extracellular signal-regulated kinase (ERK) were obtained from the Santa Cruz Co., and the anti-COX-2 antibody was acquired from Caymen Chemicals (Ann Arbor, Mich.).

Techniques: Construct, Incubation, Pull Down Assay, SDS Page, Western Blot, Staining, Infection, Recombinant, Immunoprecipitation, Transfection, Luciferase, Expressing, Labeling