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

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension.

doi: 10.1016/j.biopha.2018.11.058

Figure Lengend Snippet: Fig. 4. Effect of miR-150 on pulmonary fibrosis of pulmonary hypertension rats. (A) Pulmonary fibrosis was detected by Masson’s staining (200× magnification). Scale bars, 100 μm. (B) The area of pulmonary fibrosis was calculated and shown. The mRNA expressions of TGF-β1 (C) and collagen I (D) in lung tissues were evaluated by qPCR. (E) The protein levels of TGF-β1 and collagen I in lung tissues were measured by western blot assay. (F)&(G) Relative grey values of the protein bands were shown. (H) The expressions of TGF-β1 and collagen I in lung tissues were detected by immunohistochemical staining (400× magnification). Scale bars, 50 μm. Data were presented as mean ± SD. **P < 0.01, ***P < 0.001 versus the indicated group.

Article Snippet: The membranes were blocked in 5% nonfat milk for 1 h at room temperature, then incubated with primary antibodies against TGF-β1 (1:400, BOSTER, China), Collagen I (1:300, BOSTER, China), p-AKTser473 (1:500, KeyGen, China), AKT (1:500, KeyGen, China), p-mTORser2481 (1:500, Sangon Biotech, China), mTOR (1:1000, Cell signaling Technology, USA), and β-actin (1:500, Bioss, China) at 4°C overnight, followed by incubation with HRP-labeled Goat AntiRabbit or Goat Anti-Mouse IgG (1:5000, Beyotime, China) at 37°C for 45min.

Techniques: Staining, Western Blot, Immunohistochemical staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension.

doi: 10.1016/j.biopha.2018.11.058

Figure Lengend Snippet: Fig. 6. Effect of miR-150 on the expressions of fibrosis-related molecules. The mRNA expressions of TGF-β1 (A) and collagen I (B) in PASMCs were measured by qPCR. (C) The protein levels of TGF-β1 and collagen I in PASMCs were detected by western blot assay. (D)&(E) Relative grey values of the protein bands were shown. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group.

Article Snippet: The membranes were blocked in 5% nonfat milk for 1 h at room temperature, then incubated with primary antibodies against TGF-β1 (1:400, BOSTER, China), Collagen I (1:300, BOSTER, China), p-AKTser473 (1:500, KeyGen, China), AKT (1:500, KeyGen, China), p-mTORser2481 (1:500, Sangon Biotech, China), mTOR (1:1000, Cell signaling Technology, USA), and β-actin (1:500, Bioss, China) at 4°C overnight, followed by incubation with HRP-labeled Goat AntiRabbit or Goat Anti-Mouse IgG (1:5000, Beyotime, China) at 37°C for 45min.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Fluorofenidone Offers Improved Renoprotection at Early Interventions during the Course of Diabetic Nephropathy in db/db Mice via Multiple Pathways

doi: 10.1371/journal.pone.0111242

Figure Lengend Snippet: A) RNA was purified from the indicated mice (n = 3 per group). TGF-β1 mRNA abundance was subsequently determined by real-time PCR following our published methodology , , . Real-time PCR was performed 3 times in triplicate. TGF-β1 mRNA levels were normalized to actin, and expressed as the fold change in reference to TGF-β1 mRNA in db/m mice. *: p <0.05 in comparison to db/m ; #: p <0.05 in comparison to the respective Ctrl mice; other comparison are also presented. B) TGF-β1 protein in the indicated mice (n = 6 per group) was quantified using ELISA. Assay was repeated three times in triplicate. TGF-β1 protein was expressed as ng per mg of total protein (ng/mg total protein). Statistical analysis was performed as detailed above.

Article Snippet: Kidney-associated TGF-β1 protein abundance was measured using an ELISA kit (Boster Biological Technology, Wuhan, China) according to the manufacturers' instructions.

Techniques: Purification, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT

doi: 10.1038/srep11924

Figure Lengend Snippet: A : TGFβ1 in conditional mediums secreted by 5637 (CTRL), NF and CAF cells were quantified by ELISA. B : The mRNA expression levels of TGFβ1 in CAFs, NFs and three bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. C : The expression of phosphorylated Smad2 and total Smad2 protein in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. D : The expression levels of TGFβ1 and TGFβRII in bladder cancer cell lines cultured with CAF-CM were detected by qRT-PCR using β-actin gene as the normalization control. *** P < 0.001.

Article Snippet: The concentrations of TGFβ1 in different media were measured using human TGFβ1 ELISA kit (BOSTER, EK0513, Wuhan, China), according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Control, Western Blot, Cell Culture

Journal: Scientific Reports

Article Title: TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT

doi: 10.1038/srep11924

Figure Lengend Snippet: A : Summary of altered expression of lncRNAs in the CAF-CM-treated 5637 and J82 cells, comparing to the NF-CM-treated ones. 1.5-fold of relative mRNA level using qRT-PCR, normalized by β-actin gene, was used as the threshold for significant changes. B and C : ZEB2NAT expression levels in the CAF-CM treated 5637 and J82 cells upon the treatments of a TGFβ1 neutralizing antibody ( B ) and a TGFβRI inhibitor (SB-431542) ( C ), respectively. D : Exogenous expression of ZEB2NAT lncRNA in 5637 cells, detected by qRT-PCR using β-actin gene as the normalization control. E – G : Effects of ZEB2NAT lncRNAs overexpression on cell invasion by the Transwell invasion assay ( E , F ) and protein levels of E-cadherin, ZEB1 and ZEB2 by immunoblotting ( G ) in 5637 cells. H: Knockdown of ZEB2NAT by RNAi using two siRNAs targeting different regions of the lncRNA (siZEB2NAT-1 and siZEB2NAT-2) in 5637 cells. The relative ZEB2NAT expression levels were at 48 hours after siRNA transfection and determined by qRT-PCR using β-actin gene as the normalization control. I , J , K : Effects of ZEB2NAT lncRNAs knockdown on cell invasion by the Transwell invasion assay ( I , J ) and protein levels of E-cadherin and ZEB2 by immunoblotting ( K ) in the CAF-CM treated 5637 cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The concentrations of TGFβ1 in different media were measured using human TGFβ1 ELISA kit (BOSTER, EK0513, Wuhan, China), according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Over Expression, Transwell Invasion Assay, Western Blot, Knockdown, Transfection

Journal: Scientific Reports

Article Title: TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT

doi: 10.1038/srep11924

Figure Lengend Snippet: The expression levels of ZEB2NAT ( A ) and TGFβ1 transcripts ( B ), and ZEB2 protein ( C ) in 30 human bladder cancer tissues (Tumor or T) and the paired normal tissues (Normal or N) from the same patient. Relative ZEB2NAT and TGFβ1 mRNA levels were assessed by qRT-PCR and normalized by β-actin gene. D – F : The correlations among ZEB2NAT, TGFβ1 and ZEB2 were decided by Pearson correlation analysis. ΔCT values in qRT-PCR were used as the expression levels of ZEB2NAT and TGFβ1 transcripts. ZEB2 protein bands on Western blot were quantified by Image J software. P < 0.05 was considered statistically significant.

Article Snippet: The concentrations of TGFβ1 in different media were measured using human TGFβ1 ELISA kit (BOSTER, EK0513, Wuhan, China), according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software

Journal: Journal of advanced research

Article Title: Plasma-activated solutions prevent peritoneal adhesion formation by regulating eNOS expression in mesothelial cells.

doi: 10.1016/j.jare.2025.02.024

Figure Lengend Snippet: Fig. 2. PAS attenuates apoptosis, ROS accumulation, and extracellular matrix component deposition in the peritoneum. (A) Representative immunofluorescence staining images of TUNEL staining of peritoneum in different groups. Scale bars, 100 lm. (B) Representative immunofluorescence images showing ROS accumulation in peritoneum in different groups. Scale bars, 100 lm. (C) Representative images of Masson’s trichrome staining with different magnification (100 ×; 400×) indicating collagen deposition in the peritoneum. Scale bars, 200 lm for 100 × images. (D, E) ELISA measurements for TGF-b1 (D) and IL-6 (E) in serum of mice 7 days after adhesion induction with administration of PBS or PAS. n = 3 in each group. (F, G) Representative dual-immunofluorescence images of peritoneum (ischemic button) and/or adhesion tissues. Sections were co-stained with Collagen-I and CK19 (F) or a-SMA and CK19 (G), while CK19 and DAPI were utilized to localize peritoneal mesothelial monolayer. The fluorescence intensity of Collagen-I or a-SMA reflects the relative protein expression levels. Scale bars, 100 lm. Data are shown as mean ± SD and statistical analysis are performed with one-way ANOVA. *p < 0.05, ** p < 0.01, *** p < 0.001, n.s.: no significance.

Article Snippet: The levels of IL-6 and TGF-b1 were determined using corresponding ELISA kits (IL-6: Proteintech, KE10007; TGF-b1: Proteintech, KE10005) following the manufacturer’s standard protocols.

Techniques: Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of advanced research

Article Title: Plasma-activated solutions prevent peritoneal adhesion formation by regulating eNOS expression in mesothelial cells.

doi: 10.1016/j.jare.2025.02.024

Figure Lengend Snippet: Fig. 6. PAS attenuates apoptosis, ROS accumulation, and extracellular matrix component deposition by restoring eNOS expression in mesothelial cells. (A) Representative immunofluorescence staining images of TUNEL staining of peritoneum across four experimental groups: (i) PBS, (ii) PBS + L-NAME, (iii) PAS, and (iv) PAS + L- NAME. n = 5 in each group. Scale bars, 100 lm. (B) Representative immunofluorescence images showing ROS accumulation in peritoneum in different groups. Scale bars, 100 lm. (C) Representative images of Masson’s trichrome staining with different magnification (100 ×; 400 × ) indicating collagen deposition in the peritoneum. Scale bars, 200 lm for 100 × . (D, E) ELISA measurements for TGF-b1 (D) and IL-6 (E) in serum of mice 7 days after adhesion induction in different groups. n = 3 in each group. (F, G) Representative dual-immunofluorescence images of peritoneum (ischemic button) and/or adhesion tissues. Sections were co-stained with Collagen-I and CK19 (F) or a-SMA and CK19 (G), while CK19 and DAPI were utilized to localize peritoneal mesothelial monolayer. The fluorescence intensity of Collagen-I or a-SMA reflects the relative protein expression levels. Scale bars, 100 lm. Data are shown as mean ± SD and statistical analysis are performed with one-way ANOVA. *p < 0.05, ** p < 0.01, *** p < 0.001, n.s.: no significance.

Article Snippet: The levels of IL-6 and TGF-b1 were determined using corresponding ELISA kits (IL-6: Proteintech, KE10007; TGF-b1: Proteintech, KE10005) following the manufacturer’s standard protocols.

Techniques: Expressing, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: CD168 + macrophages promote hepatocellular carcinoma tumor stemness and progression through TOP2A /β-catenin/ YAP1 axis

doi: 10.1016/j.isci.2023.106862

Figure Lengend Snippet:

Article Snippet: Human TGF-beta1 ELISA Kit , proteintech , Cat # KE00002.

Techniques: Recombinant, Preserving, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Software