Journal: Materials Today Bio

Article Title: Uniform and controllable surface nano-structure on polyetheretherketone implants can regulate mechanical property to enhance soft tissue integration through Piezo1/TGF-β1 signaling axis

doi: 10.1016/j.mtbio.2025.101645

Figure Lengend Snippet: The impact of nanoscale modification of PEEK implant surfaces on the mechanical perception, proliferation, and expression of fibrosis-related proteins in L929 cells. (A) The expression levels of mechanical perception, proliferation, and fibrosis-related proteins were evaluated in L929 cells cultured on different nanoscale modified PEEK surfaces at multiple time points. (B) The secretion of TGF-β1 from L929 cells cultured on various nanoparticle-modified PEEK surfaces was quantified using Elisa at multiple time points. (C) Immunofluorescence analysis was conducted to assess the expression differences of Piezo1, ITGB1, and α-SMA in L929 cells cultured on different nanoparticle-modified PEEK surfaces. (D) Quantitative fluorescence analysis was performed on the data presented in C.

Article Snippet: TGF-β1 Mouse/Rat (HEK293) (HY-P70648, MedChemExpress LLC., USA).

Techniques: Modification, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence

Journal: Materials Today Bio

Article Title: Uniform and controllable surface nano-structure on polyetheretherketone implants can regulate mechanical property to enhance soft tissue integration through Piezo1/TGF-β1 signaling axis

doi: 10.1016/j.mtbio.2025.101645

Figure Lengend Snippet: Piezo1 mediates the proliferation and fibrosis enhancement of L929 cells on PEEK implants modified with a 500 nm surface treatment. (A) Changes in calcium influx induced by the specific Piezo1 agonist (Yoda1) and the calcium channel inhibitor (GsMTx-4) were assessed using Fluo-4AM. (B) The fluorescence intensity of calcium accumulation in L929 cells following treatment with Yoda1 and GsMTx-4 was observed under a fluorescence microscope. (C) Quantitative analysis of the average fluorescence intensity presented in B. (D) The regulatory effects of Piezo1 on proliferation-related pathways, fibrosis processes, and adhesion-associated proteins were evaluated separately on PEEK and PEEK-500. (E) RT-qPCR was employed to assess the transcriptional differences in fibrosis-related genes in L929 cells following the activation or inhibition of Piezo1. (F) Changes in TGF-β1 secretion by L929 cells after the activation or inhibition of Piezo1 were analyzed. (G) The involvement of TGF-β1 in fibrosis regulation through the activation and upregulation of Piezo1 was confirmed. (H) Schematic representation of the mechanism by which L929 cells detect mechanical signals from PEEK samples to facilitate soft tissue integration.

Article Snippet: TGF-β1 Mouse/Rat (HEK293) (HY-P70648, MedChemExpress LLC., USA).

Techniques: Modification, Fluorescence, Microscopy, Quantitative RT-PCR, Activation Assay, Inhibition

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: 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: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A and B ) HEK293T cells and MDA-MB-231 cells were serum starved and treated with TGFB1 (5 ng/mL) for the indicated durations, and whole-cell extracts (WCEs) were collected for IP with anti-SMAD3 antibody, followed by immunoblot (IB) analysis. ( C ) HEK293T cells were transfected with WT HA - SMAD3 or mutant plasmids as indicated and treated with TGFB1 (5 ng/mL). WCEs were then collected for IP with anti-HA antibody, followed by IB analysis. ( D ) SMAD3 K53/K333 site aa in different species. ( E ) HEK293T cells were transfected with WT HA - SMAD3 or K53/333R-mutant plasmids and then treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-HA antibody, followed by IB analysis. ( F ) ddH 2 O (10 μL) containing different peptides (0.1–0.75 μg) was added onto the PVDF membranes, followed by IB analysis using a K53-specific trimethylation antibody (anti–SMAD3 K53me3) and a K333-specific trimethylation antibody (anti–SMAD3 K333me3). ( G ) MDA-MB-231 cells were serum starved and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis with SMAD3 K53/K333 trimethylation–specific antibodies. ( H ) MDA-MB-231 SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Western Blot, Transfection, Mutagenesis, Stable Transfection

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) MDA-MB-231 SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). Quantitative RT-PCR analysis of TGFB / SMAD3 signaling pathway downstream genes, including CTGF , PAI1 , PDGFB , and SMAD7 , in the indicated cells with or without TGFB1 (5 ng/mL) treatment. ( B ) Quantitative analysis of Transwell assay in the indicated cells. ( C ) IF and ( D ) IB analysis of EMT markers in the indicated cells. Scale bar: 50 um. ( E and F ) Representative lung image ( E ) and H&E-stained lung sections ( F ). Scale bars: 5 mm. ( G and H ) Scatter plots showing lung metastatic nodules ( G ) and lung weights ( H ). All immunoblots were performed 3 times, independently, with similar results. Data indicate the mean ± SD. ** P < 0.05, by 2-tailed Student’s t test ( A , B , G , and H ).

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Stable Transfection, Transfection, Quantitative RT-PCR, Transwell Assay, Staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) WCEs of MDA-MB-231 and MCF-7 cells were collected and subjected to co-IP and IB assays. ( B ) HEK293T and MCF-7 cells were serum starved and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( C ) MDA-MB-231 and MCF-7 cells were treated with TGFB1 (5 ng/mL) and the EZH2 inhibitors GSK126 or GSK503, and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( D ) HEK293T cells were transfected with WT HA-SMAD3 or mutant plasmids and a Flag-EZH2 plasmid as indicated/WCEs were then collected for IP with anti-HA antibody, followed by IB analysis. ( E ) Immunoprecipitated EZH2 from HEK293 cells was incubated with SAM along with SMAD3 protein for in vitro methylation of SMAD3 . The methylated proteins were separated by SDS-PAGE, and SMAD3 methylation was analyzed by IB using anti–SMAD3 K53/K333 trimethylation–specific antibodies. ( F ) MDA-MB-231 cells silenced with control (shNC) or EZH2 shRNA (nos. 1 and 2) were treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( G ) HEK293T cells were transfected with vector, EZH2 WT , or EZH2 H689A and then treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( H ) HEK293T cells were transfected with vector, EZH2 WT , or EZH2 Y641H , and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Incubation, In Vitro, Methylation, SDS Page, Control, shRNA, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) Quantitative analysis of Transwell assay in the indicated MDA-MB-231 cells treated with TGFB1 (5 ng/mL). ( B ) MDA-MB-231 SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and silenced with control or EZH2 shRNA (nos. 1 and 2). WCEs were collected for IB analysis. ( C ) WT Flag - EZH2 or a Flag - EZH2 H689A plasmid was transfected into MDA-MB-231 SMAD3–/– cells ectopically expressing WT SMAD3 or SMAD3 K53/333R, and WCEs were collected for IB analysis. ( D and E ) A Transwell cell invasion assay was performed using MDA-MB-231 SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and transfected with a vector, EZH2 WT , or EZH2 H689A . Representative images ( D ) and quantitative analysis ( E ). Original magnification, ×200. ( F – H ). Representative lung image ( F ),H&E-stained lung sections ( G ), and scatter plot showing lung weights ( H ). Scale bars: 5 mm. All immunoblotting was performed 3 times, independently, with similar results. Data indicate the mean ± SD. ** P < 0.05, by 2-tailed Student’s t test ( A , E , and H ).

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Transwell Assay, Stable Transfection, Transfection, Control, shRNA, Plasmid Preparation, Expressing, Invasion Assay, Staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) Membrane and cytosolic fractions from MDA-MB-231 SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids were collected and subjected to IB analysis. ( B ) Membrane fractions from MDA-MB-231 SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL) were collected and subjected to IB analysis. ( C ) MDA-MB-231 SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). IF images show the cellular localization of SMAD3 . Scale bar: 10 μm. ( D ) Membrane and cytosolic fractions from MDA-MB-231 cells silenced with control or EZH2 shRNA (no. 1) were collected and subjected to IB analysis. ( E ) HEK293T cells were transfected with WT HA- SMAD3 or HA- SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-HA antibody, followed by IB analysis. ( F ) HEK293T cells were silenced with control or EZH2 shRNA (nos. 1 and 2), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( G ) Co-IP of endogenous SMAD3 from MDA-MB-231 cells transfected with EZH2 WT or EZH2 H689A , followed by IB analysis. ( H ) Co-IP of endogenous SMAD3 from MDA-MB-231 cells transfected with EZH2 WT or EHZ2 Y641H , followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Membrane, Stable Transfection, Transfection, Control, shRNA, Co-Immunoprecipitation Assay, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) The aa sequence of different TAT peptides. ( B and C ) HEK293T cells were treated with different TAT peptides (Pep-1, Pep-2) and TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( D ) MDA-MB-231 were silenced with TAT peptides and treated with TGFB1 (5 ng/mL), and WCEs were collected for IB analysis. ( E ) Quantitative analysis of Transwell cell migration and invasion assays using MDA-MB-231 cells treated with different TAT peptides. ( F and G ) Representative lung image ( F ) and H&E-stained lung sections ( G ). Scale bars: 10 mm. ( H and I ) Scatter plots show the number of lung metastatic nodes ( I ) and lung weights ( H ). All immunoblotting was performed 3 times, independently, with similar results. Data indicate the mean ± SD. ** P < 0.05, by 2-tailed Student’s t test.

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Sequencing, Migration, Staining, Western Blot