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: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: TGF-β1 promotes the osteoblastic differentiation of MC3T3-E1 cells. (A) RT-qPCR analysis of the osteogenic-related genes RUNX2, OSX and COL1A1 in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (B) Western blotting of RUNX2, COL1A1-and OSX protein in MC3T3-E1 cells cultured with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (C) ALP activity in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. NS, no significance change; *P<0.05, **P<0.01 as indicated. TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; OSX, osterix; COL1A1 collagen type I α1 chain; ALP, alkaline phosphatase.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (cat. no. CSB-AP003861HU; CUSABIO Technology LLC, Wuhan, China) with final concentration of 4 ng/ml was added to cell culture for 7 or 14 days.

Techniques: Quantitative RT-PCR, Western Blot, Cell Culture, Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: miR-877-3p promotes the osteoblastic differentiation of MC3T3-E1 cells. (A) miR-877-3p expression in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (B) Expression of miR-877-3p was validated in MC3T3-E1 cells transfected with miR-877-3p mimics or inhibitor and their corresponding controls by RT-qPCR. (C) RUNX2, OSX and COL1A1 mRNA expression in MC3T3-E1 cells. (D) MC3T3-E1 cells transfected with miR-877-3p mimics or inhibitor and their corresponding controls were cultured in osteogenic medium. At day 14, the mineralization of differentiated MC3T3-E1 cells was detected with ARS and ALP staining. (E) Transfected MC3T3-E1 cells were cultured in osteogenic medium for 14 days and then mixed with tricalcium phosphate/hydroxyapatite and transplanted into the dorsal region of nude mice for 4-weeks. The results were then evaluated by H&E and Masson's trichrome staining. (F) (F) Transfected MC3T3-E1 cells were cultured in osteogenic medium for 14 days and then mixed with tricalcium phosphate/hydroxyapatite and transplanted into the dorsal region of nude mice for 4-weeks. The Expression expression of miR-877-3p in the xenografts from each group was detected by RT-qPCR analysis. *P<0.05, **P<0.01 and ***P<0.001, as indicated. miR, microRNA; TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; OSX, osterix; COL1A1 collagen type I α1 chain; NC, negative control; ARS, Alizarin Red S; ALP, alkaline phosphatase; H&E, hematoxylin and eosin.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (cat. no. CSB-AP003861HU; CUSABIO Technology LLC, Wuhan, China) with final concentration of 4 ng/ml was added to cell culture for 7 or 14 days.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Cell Culture, Staining, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: miR-877-3p inhibits the expression of Smad7. (A) Design of luciferase reporters with the WT Smad7 3′UTR or the site-directed MUT Smad7 3′UTR. (B) Effect of miR-877-3p and miR-NC on luciferase activity in MC3T3-E1 cells transfected with the WT or MUT Smad7 3′UTR. Smad7 expression detected by (C) RT-qPCR and (D) western blotting in MC3T3-E1 cells after culture with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. Smad7 (E) mRNA and (F) protein expression in MC3T3-E1 cells transfected with miR-877-3p mimics or inhibitors and their corresponding controls. *P<0.05, **P<0.01 as indicated. miR, microRNA; WT, wild type; MUT, mutant; UTR, untranslated region; NC, negative control; TGF, transforming growth factor.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (cat. no. CSB-AP003861HU; CUSABIO Technology LLC, Wuhan, China) with final concentration of 4 ng/ml was added to cell culture for 7 or 14 days.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Mutagenesis, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: Smad7 is identified as a downstream target of miR-877-3p during the osteoblastic differentiation of MC3T3-E1 cells. mRNA expression of (A) Smad7 and (B) osteoblast differentiation-associated genes in MC3T3-E1 cells transfected with inhibitor-NC, miR-877-3p inhibitor or miR-877-3p inhibitor plus TGF-β1. (C) Expression of RUNX2, Smad7, p-Smad2, t-Smad2, p-Smad3 and t-Smad3 protein in MC3T3-E1 cells transfected with inhibitor-NC, miR-877-3p inhibitor or miR-877-3p inhibitor plus TGF-β1. (D) The mRNA expression of Smad7 was validated in MC3T3-E1 cells transfected with pcDNA-NC or pcDNA-Smad7. The mRNA expression of (E) Smad7 and (F) osteoblast differentiation-associated genes in MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7. (G) Protein levels of Smad7, RUNX2, p-Smad2, t-Smad2, p-Smad3 and t-Smad3 in MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7. (H) ALP activity of MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7 after culture with osteogenic medium for 7 days. (I) MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7 were induced with osteogenic medium. At day 14, the mineralization of differentiated MC3T3-E1 cells was detected with ARS and ALP staining. *P<0.05, **P<0.01 as indicated. miR, microRNA; NC, negative control; TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; p, phospho; t, total; ALP, alkaline phosphatase; ARS, Alizarin Red S.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (cat. no. CSB-AP003861HU; CUSABIO Technology LLC, Wuhan, China) with final concentration of 4 ng/ml was added to cell culture for 7 or 14 days.

Techniques: Expressing, Transfection, Activity Assay, Staining, Negative Control