tgf-β1 Search Results


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  • 94
    Millipore tgf β1
    NEF is likely an upstream inhibitor of <t>TGF-β1</t> in glioma cells. (A) NEF expression and (B) TGF-β1 expression in non-transfected cells, cells transfected with empty vectors and cells transfected with NEF overexpressing vectors from two human glioma cell lines. (C) NEF expression in Hs 683 cells following treatment with 5, 10, 20 and 30 ng/ml TGF-β1. C, control; NC, negative control; TGF, transforming growth factor. *P
    Tgf β1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human tgf β1
    TGRL lipolysis products release <t>TGF-β1</t> at 15 min. The rate of TGF-β1 release is significantly increased for cells treated with TGRL (150 mg/dL) + LpL (2 U/mL) (TL) compared to cells treated with Media (M) or LpL alone (L) or TGRL alone (T), at 15 min. Addition of 10 μM of ALK to TL (TL+ALK) suppressed TGF-β1 released by TL. N = 4/treatment group, P ≤0.05 as significant, * = TL compared to M, L, T or TL, # = TL+ALK compared to TL. TGF-β1 was not detected in M, T or TL only, in the absence of cells.
    Recombinant Human Tgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 2249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam tgf β1
    Increased expression of Numb in human fibrotic kidney A. Representative micrographs show the collagen deposition detected by Masson trichrome staining in human normal and fibrotic kidneys. Bar=50μm. B. Representative images of the immunohistochemical staining of Numb, <t>TGF-β1</t> and p-H3 in sequential sections of renal biopsies from patients with renal tubulointerstitial fibrosis. Normal renal tissues adjacent to tumor (renal cell carcinoma) were used as normal control. Arrows indicate the co-localization of Numb, TGF-β1 and p-H3 in the injured tubules. Bar=50μm.
    Tgf β1, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 1777 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology tgf β1
    rLRRFIP-1 treatment affects <t>TGF-β</t> levels and early collagen deposition in wounds in vivo. ( A , B ) Representative images and graphical analysis of <t>TGF-β1</t> and TGF-β3 levels (white fluorescence) in day 3 and day 7 wounds treated with rLRRFIP-1 (1 µg/mL) or PBS control illustrating decreased TGF-β1 and increased TGF-β3 levels in wounds in vivo following rLRRFIP-1 treatment. Epidermis is delineated by white dotted lines, e denotes epidermis, and w denotes wound dermis. Magnification ×20. Scale Bar = 50 µm; ( C , D ) Representative images and graphical analysis of total collagen levels in day 7 wounds in vivo post-treatment with rLRRFIP-1 (1 µg/mL) or PBS control illustrating increased total collagen deposition in wounds (green) following treatment with rLRRFIP-1 (1 µg/mL). Magnification ×10. Scale Bar = 100 µm. Results represent mean ± SEM. * p
    Tgf β1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PeproTech recombinant human tgf β1
    Changes of IgA CSR in PP and LP following intestinal I/R. ( A ) Changes of <t>TGF</t> <t>‐β1</t> and AID in PP and LP at 5 min. and 2 hrs after reperfusion. Analysis of relative mRNA expression of these two biomarkers was shown, respectively. And representative blots were presented on the top. ( B ) Changes of GLT α and α CT s in PP and LP at 5 min. and 2 hrs after reperfusion. ( C ) Changes of IgA + B220 + and IgM + B220 + cells from PP and LP at 2 hrs after reperfusion. Representative flow cytometric profiles were presented in the left panel. Analysis of the percentage of IgA + and IgM + B220 + cells was shown in the right. Data are expressed as mean ± S.E., n = 5 or 6. Results were compared by anova with Tukey post‐test. $ P
    Recombinant Human Tgf β1, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 1496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech tgf β1
    <t>TGF-β1–mediated</t> migration is reduced in Itga3 –/– keratinocytes.
    Tgf β1, supplied by PeproTech, used in various techniques. Bioz Stars score: 95/100, based on 3147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc tgf β1
    Enrichment of miR-744 reduced phosphorylation of Smad3 and increased the production of SARA in epithelial cells from normal or severe asthma subject. ( A ) Western blot analysis was conducted to detected total and phosphorylated Smad3 or SARA expression in NBECs and SABECs with <t>TGF-β1</t> overexpression vector or (and) miR-744 mimic or miR-NC transfection. ( B–D ) Relative densitometry analyses of proteins were conducted using Image Lab software. * P
    Tgf β1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 654 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human tgfβ1
    TNF-α suppresses <t>TGFβ1-dependent</t> production of SMαA protein and mRNA in human pulmonary myofibroblasts. (a) hPFBs were pretreated with TNF-α (10 ng/ml) for 2 h followed by exposure to TGFβ1 (5 ng/ml) for another
    Recombinant Human Tgfβ1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 862 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher tgf β1
    Effect of <t>TGF-β1</t> on Smad3 deficient versus WT mast cell proliferation
    Tgf β1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson tgf β1
    Overexpression of circPTK2 enhances TIF1γ expression and inhibits <t>TGF-β-induced</t> EMT and invasion of NSCLC cells in vitro. a TIF1γ mRNA and protein levels in A549 and H226 cells transiently overexpressing circPTK2. Relative TIF1γ expression was determined with normalization against β-actin. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing circPTK2 were treated with or without <t>TGF-β1</t> (5 ng/ml) for 1 h and 0.5 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. c A549 and H226 cells transiently overexpressing circPTK2 were serum-starved for 24 h and then treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Snail and N-cadherin protein levels were determined by western blot. β-actin was used as internal control. d , e A549 and H226 cells transiently overexpressing circPTK2 were treated as above and subjected to the transwell migration and invasion assays. Migrated and invasive cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. * P
    Tgf β1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 531 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti tgf β1
    Co-culture of macrophages and myofibroblasts induces α-SMA, <t>TGF-β1</t> and Smad3 expression. ( A ) Representative immunohistochemical staining of α-SMA with 3-D co-culture in the present of Ang II. Scale bars: 50 µm. ( B ) Real-time PCR quantification of α-SMA and TGF-β1 mRNA expression. ( C ) Immunofluorescence staining of protein level of phosphorylated Smad3 (p-Smad3; green) and DDR2 (red) or Immunofluorescence staining of TGF-β1 (green) and DDR2 (red) in co-cultured WT macrophages and fibroblasts with neutralizing antibody to IL-6 and co-cultured IL-6-/- macrophages and fibroblasts with recombinant IL-6 (rIL-6) treatment. Scale bars: 50 µm. (n = 6 per group). ( D ) Western blot analysis and quantification of protein level of TGF-β1 and p-Smad3 in 3-D co-culture (n = 4 per group). * P
    Anti Tgf β1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti tgf β1
    HGF decreases α-SMA expression in HUVECs through <t>TGF-β/Smad</t> and Akt/mTOR/p70S6K signaling pathways. ( A, B ) HUVECs treated with 40 ng/mL HGF and/or 5 ng/mL <t>TGF-β1</t> for 48 hours. We collected equal amounts of protein from whole cell lysates, which we analyzed by western blotting with antibodies against phosphorylated Smad 2, Smad 3, Akt, mTOR, p70 S6K, Erk1/2, c-Jun. ( C, D ) HUVECs were pretreated for one hour with MK2206 (10 μmol/L) ( C ), and SB431542 (10 μmol/L) ( D ), which are specific chemical inhibitors for Akt and Smad, respectively. Subsequently, we treated cells with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours. We collected cells one hour after HGF and/or TGF-β1 stimulation. We analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against phosphorylated and total Akt ( C ), phosphorylated and total mTOR ( C ), phosphorylated and total p70S6K ( C ), phosphorylated and total Smad 2 ( D ), phosphorylated and total Smad 3 ( D ). ( C, D ) We collected HUVECs 48 hours after HGF and/or TGF-β1 stimulation and analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against α-SMA and GAPDH.
    Anti Tgf β1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    R&D Systems tgf β 1
    The effects of actin microfilament inhibition on basal and <t>TGF</t> β 1-stimulated superficial zone protein (SZP) media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 1, 6, and 10 μM cytochalasin
    Tgf β 1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 722 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human tgf beta 1 protein
    The effects of actin microfilament inhibition on basal and <t>TGF</t> β 1-stimulated superficial zone protein (SZP) media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 1, 6, and 10 μM cytochalasin
    Recombinant Human Tgf Beta 1 Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 98/100, based on 367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti tgf beta 1 antibody
    The effects of actin microfilament inhibition on basal and <t>TGF</t> β 1-stimulated superficial zone protein (SZP) media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 1, 6, and 10 μM cytochalasin
    Anti Tgf Beta 1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad tgf β1
    Immunoblot analysis of the expression of <t>TGF-β1</t> and VEGF after 7, 14 and 21 days of the experiment in second-degree burns in rats in different treatment groups. C: untreated control; L: treated with 670-nm InGaP laser; P: treated with the Porophyllum ruderale extract PL: treated with the P. ruderale extract and 670-nm InGaP laser. Typical blots are shown above average densitometry results. Values are the mean and standard deviation of each group and were compared by ANOVA with Tukey’s post-test (* p
    Tgf β1, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    PeproTech recombinant tgf β1
    NMuMG cells respond to both <t>TGF-β3</t> WW and TGF-β3 WD . NMuMG cells were untreated (Un) or treated with <t>TGF-β1</t> (T), BMP4 (B) or the indicated concentrations of TGF-β3 WW and TGF-β3 WD for 1 hr. Whole cell lysates were immunoblotted with the antibodies shown. Both TGF-β3 WW and TGF-β3 WD induce phosphorylation of pSMAD1/5, although the latter is less potent.
    Recombinant Tgf β1, supplied by PeproTech, used in various techniques. Bioz Stars score: 98/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    NEF is likely an upstream inhibitor of TGF-β1 in glioma cells. (A) NEF expression and (B) TGF-β1 expression in non-transfected cells, cells transfected with empty vectors and cells transfected with NEF overexpressing vectors from two human glioma cell lines. (C) NEF expression in Hs 683 cells following treatment with 5, 10, 20 and 30 ng/ml TGF-β1. C, control; NC, negative control; TGF, transforming growth factor. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: lncRNA NEF inhibits glioma by downregulating TGF-β1

    doi: 10.3892/etm.2019.7602

    Figure Lengend Snippet: NEF is likely an upstream inhibitor of TGF-β1 in glioma cells. (A) NEF expression and (B) TGF-β1 expression in non-transfected cells, cells transfected with empty vectors and cells transfected with NEF overexpressing vectors from two human glioma cell lines. (C) NEF expression in Hs 683 cells following treatment with 5, 10, 20 and 30 ng/ml TGF-β1. C, control; NC, negative control; TGF, transforming growth factor. *P

    Article Snippet: An ELISA kit was used to measure blood levels of TGF-β1 (cat. no. RAB0460-1KT; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol.

    Techniques: Expressing, Transfection, Negative Control

    NEF overexpression promotes the migration and invasion of glioma cells. (A) Migration and (B) invasion ability of non-transfected cells, cells transfected with empty vectors, NEF overexpressing vectors or exogenous TGF-β1, and cells co-transfected with NEF overexpressing vectors and exogenous TGF-β1 from two human glioma cell lines. C, control; NC, negative control; TGF, transforming growth factor. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: lncRNA NEF inhibits glioma by downregulating TGF-β1

    doi: 10.3892/etm.2019.7602

    Figure Lengend Snippet: NEF overexpression promotes the migration and invasion of glioma cells. (A) Migration and (B) invasion ability of non-transfected cells, cells transfected with empty vectors, NEF overexpressing vectors or exogenous TGF-β1, and cells co-transfected with NEF overexpressing vectors and exogenous TGF-β1 from two human glioma cell lines. C, control; NC, negative control; TGF, transforming growth factor. *P

    Article Snippet: An ELISA kit was used to measure blood levels of TGF-β1 (cat. no. RAB0460-1KT; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol.

    Techniques: Over Expression, Migration, Transfection, Negative Control

    Blood levels of NEF are significantly correlated with TGF-β1 in patients with metastatic glioma. Pearson's correlation coefficient analysis of correlations between blood NEF and TGF-β1 levels in (A) patients with metastatic glioma, (B) patients with non-metastatic glioma and (C) healthy controls. TGF, transforming growth factor.

    Journal: Experimental and Therapeutic Medicine

    Article Title: lncRNA NEF inhibits glioma by downregulating TGF-β1

    doi: 10.3892/etm.2019.7602

    Figure Lengend Snippet: Blood levels of NEF are significantly correlated with TGF-β1 in patients with metastatic glioma. Pearson's correlation coefficient analysis of correlations between blood NEF and TGF-β1 levels in (A) patients with metastatic glioma, (B) patients with non-metastatic glioma and (C) healthy controls. TGF, transforming growth factor.

    Article Snippet: An ELISA kit was used to measure blood levels of TGF-β1 (cat. no. RAB0460-1KT; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol.

    Techniques:

    TGRL lipolysis products release TGF-β1 at 15 min. The rate of TGF-β1 release is significantly increased for cells treated with TGRL (150 mg/dL) + LpL (2 U/mL) (TL) compared to cells treated with Media (M) or LpL alone (L) or TGRL alone (T), at 15 min. Addition of 10 μM of ALK to TL (TL+ALK) suppressed TGF-β1 released by TL. N = 4/treatment group, P ≤0.05 as significant, * = TL compared to M, L, T or TL, # = TL+ALK compared to TL. TGF-β1 was not detected in M, T or TL only, in the absence of cells.

    Journal: PLoS ONE

    Article Title: TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells

    doi: 10.1371/journal.pone.0145523

    Figure Lengend Snippet: TGRL lipolysis products release TGF-β1 at 15 min. The rate of TGF-β1 release is significantly increased for cells treated with TGRL (150 mg/dL) + LpL (2 U/mL) (TL) compared to cells treated with Media (M) or LpL alone (L) or TGRL alone (T), at 15 min. Addition of 10 μM of ALK to TL (TL+ALK) suppressed TGF-β1 released by TL. N = 4/treatment group, P ≤0.05 as significant, * = TL compared to M, L, T or TL, # = TL+ALK compared to TL. TGF-β1 was not detected in M, T or TL only, in the absence of cells.

    Article Snippet: Human TGF-β1 (100B) and recombinant human TGF-β1 (anti TGF-β1 antibody) (MAB240) from R & D Systems Inc., Minneapolis, MN.

    Techniques:

    TGRL lipolysis products activate stress response signaling via TGF-β/SMAD Signaling Pathway. Lipolysis release TGF-β1 and activate phosphorylation of Smad2 and translocation of Smad4 to nucleus. TG-β1 also activate non-Smad signaling pathways ATF3-JNK transcription factor networks. Both Smad and ATF3 further induced pro-inflammatory cytokines and apoptosis which can be inhibited by TGF-β receptor inhibitor, ALK.

    Journal: PLoS ONE

    Article Title: TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells

    doi: 10.1371/journal.pone.0145523

    Figure Lengend Snippet: TGRL lipolysis products activate stress response signaling via TGF-β/SMAD Signaling Pathway. Lipolysis release TGF-β1 and activate phosphorylation of Smad2 and translocation of Smad4 to nucleus. TG-β1 also activate non-Smad signaling pathways ATF3-JNK transcription factor networks. Both Smad and ATF3 further induced pro-inflammatory cytokines and apoptosis which can be inhibited by TGF-β receptor inhibitor, ALK.

    Article Snippet: Human TGF-β1 (100B) and recombinant human TGF-β1 (anti TGF-β1 antibody) (MAB240) from R & D Systems Inc., Minneapolis, MN.

    Techniques: Translocation Assay

    The effect of the ALK 4, 5 and 7 inhibitor or anti TGF-β antibody on the TGRL lipolysis induced ATF3 expression. HAEC were exposed to TGRL (T), TGRL lipolysis products (TL) or 20 ng/mL human TGF-β1 for 3 h. TGF-β receptor inhibitor, ALK significantly suppressed: A) mRNA expression of ATF3. N = 3, P ≤0.05. * = TL compare to T, # = TL with 10 μM of inhibitor ALK (TL+ALK) compared to TL. B) Western blot (a) and densitometry quantification (b) for ATF3 protein. N = 3, P ≤0.05. * = TL compare to T, # = TL+ALK compared to TL. TL+CD36 antibody as control (positive/negative). C) Immunofluorescence images showing nucleus accumulation of ATF3. N = 3 coverslips/treatment group, Bar = 20 μm. D) % Translocation of ATF3. N = 6 coverslips/treatment group, P ≤0.05, * = TL or TGF-β1 compare to T, # = TL+ALK compare to TL, Bar = 20 μm. anti TGF-β1 antibody (Ab) suppressed: E) mRNA expression of ATF3 was significantly suppressed. N = 3, P ≤0.05. * = TL compare to M, # = TL+ anti TGF-β1 antibody compared to TL. F) Western blot (a) and densitometry quantification (b) ATF3 protein expression was trend toward suppressed significant. N = 3, P ≤0.05. * = TL or TL + anti TGF-β1 (TLA) compare to M. Ab = anti TGF-β1 antibody.

    Journal: PLoS ONE

    Article Title: TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells

    doi: 10.1371/journal.pone.0145523

    Figure Lengend Snippet: The effect of the ALK 4, 5 and 7 inhibitor or anti TGF-β antibody on the TGRL lipolysis induced ATF3 expression. HAEC were exposed to TGRL (T), TGRL lipolysis products (TL) or 20 ng/mL human TGF-β1 for 3 h. TGF-β receptor inhibitor, ALK significantly suppressed: A) mRNA expression of ATF3. N = 3, P ≤0.05. * = TL compare to T, # = TL with 10 μM of inhibitor ALK (TL+ALK) compared to TL. B) Western blot (a) and densitometry quantification (b) for ATF3 protein. N = 3, P ≤0.05. * = TL compare to T, # = TL+ALK compared to TL. TL+CD36 antibody as control (positive/negative). C) Immunofluorescence images showing nucleus accumulation of ATF3. N = 3 coverslips/treatment group, Bar = 20 μm. D) % Translocation of ATF3. N = 6 coverslips/treatment group, P ≤0.05, * = TL or TGF-β1 compare to T, # = TL+ALK compare to TL, Bar = 20 μm. anti TGF-β1 antibody (Ab) suppressed: E) mRNA expression of ATF3 was significantly suppressed. N = 3, P ≤0.05. * = TL compare to M, # = TL+ anti TGF-β1 antibody compared to TL. F) Western blot (a) and densitometry quantification (b) ATF3 protein expression was trend toward suppressed significant. N = 3, P ≤0.05. * = TL or TL + anti TGF-β1 (TLA) compare to M. Ab = anti TGF-β1 antibody.

    Article Snippet: Human TGF-β1 (100B) and recombinant human TGF-β1 (anti TGF-β1 antibody) (MAB240) from R & D Systems Inc., Minneapolis, MN.

    Techniques: Expressing, Western Blot, Immunofluorescence, Translocation Assay

    TGF-β receptor inhibitor or anti-TGF-β1 suppressed TGRL lipolysis products-induced pro-inflammatory gene expression. Treatment with lipolysis products increased pro-inflammatory gene expression at 3 h. Effect of 10 μM of inhibitor ALK (TL+ALK): A) mRNA expression of E-selectin, IL-8 and JunB expression was significantly suppressed by TGF-β receptor inhibitor. N = 3, P ≤0.05. * = TL compare to T, # = TL+ALK compared to TL. B) mRNA expression of IL-6, NFKBIA/IκBA and NFKB1/NFκB (p50)expression was also significantly suppressed by inhibitor. N = 3, P ≤0.05. * = TL compare to T, # = TL+ALK compared to TL. Effect of 4 μg/mL of anti TGF-β1 antibody: C) mRNA expression of E-selectin and IL-8 expression was significantly suppressed by anti TGF-β1 but not JunB. N = 3, P ≤0.05. * = TL compare to M, # = TL + anti TGF-β1 compared to TL.

    Journal: PLoS ONE

    Article Title: TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells

    doi: 10.1371/journal.pone.0145523

    Figure Lengend Snippet: TGF-β receptor inhibitor or anti-TGF-β1 suppressed TGRL lipolysis products-induced pro-inflammatory gene expression. Treatment with lipolysis products increased pro-inflammatory gene expression at 3 h. Effect of 10 μM of inhibitor ALK (TL+ALK): A) mRNA expression of E-selectin, IL-8 and JunB expression was significantly suppressed by TGF-β receptor inhibitor. N = 3, P ≤0.05. * = TL compare to T, # = TL+ALK compared to TL. B) mRNA expression of IL-6, NFKBIA/IκBA and NFKB1/NFκB (p50)expression was also significantly suppressed by inhibitor. N = 3, P ≤0.05. * = TL compare to T, # = TL+ALK compared to TL. Effect of 4 μg/mL of anti TGF-β1 antibody: C) mRNA expression of E-selectin and IL-8 expression was significantly suppressed by anti TGF-β1 but not JunB. N = 3, P ≤0.05. * = TL compare to M, # = TL + anti TGF-β1 compared to TL.

    Article Snippet: Human TGF-β1 (100B) and recombinant human TGF-β1 (anti TGF-β1 antibody) (MAB240) from R & D Systems Inc., Minneapolis, MN.

    Techniques: Expressing

    The effect of the ALK 4, 5 and 7 inhibitor or anti TGF-β antibody on the TGRL lipolysis activated phosphorylation of c-Jun protein expression. HAEC were exposed to TGRL (T), TGRL lipolysis products (TL) or TL with 10 μM of inhibitor ALK (TL+ALK) for 3 h. TGF-β receptor inhibitor, ALK significantly suppressed: A) Western blot (a) and densitometry quantification (b) for p-c-Jun protein expression. N = 3, P ≤0.05. * = TL compare to T, # = TL+ALK compared to TL. B) Immunofluorescence images showing nuclear translocation of p-c-Jun. N = 3, Bar = 20 μm. anti TGF-β1 antibody (Ab) suppressed: C) Western blot (a) and densitometry quantification (b) p-c-Jun protein expression was trend toward suppressed significant. N = 3, P ≤0.05. * = TL or TL + anti TGF-β1 (TLA) compare to M.

    Journal: PLoS ONE

    Article Title: TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells

    doi: 10.1371/journal.pone.0145523

    Figure Lengend Snippet: The effect of the ALK 4, 5 and 7 inhibitor or anti TGF-β antibody on the TGRL lipolysis activated phosphorylation of c-Jun protein expression. HAEC were exposed to TGRL (T), TGRL lipolysis products (TL) or TL with 10 μM of inhibitor ALK (TL+ALK) for 3 h. TGF-β receptor inhibitor, ALK significantly suppressed: A) Western blot (a) and densitometry quantification (b) for p-c-Jun protein expression. N = 3, P ≤0.05. * = TL compare to T, # = TL+ALK compared to TL. B) Immunofluorescence images showing nuclear translocation of p-c-Jun. N = 3, Bar = 20 μm. anti TGF-β1 antibody (Ab) suppressed: C) Western blot (a) and densitometry quantification (b) p-c-Jun protein expression was trend toward suppressed significant. N = 3, P ≤0.05. * = TL or TL + anti TGF-β1 (TLA) compare to M.

    Article Snippet: Human TGF-β1 (100B) and recombinant human TGF-β1 (anti TGF-β1 antibody) (MAB240) from R & D Systems Inc., Minneapolis, MN.

    Techniques: Expressing, Western Blot, Immunofluorescence, Translocation Assay

    Smad2 phosphorylation at 1.5 h and Smad4 nucleus accumulation at 3 h by lipolysis products. A ) The nuclear isolated phosphorylated Smad2 was only observed with cellular exposure of lipolysis products (TL) or TGF-β1, not with Media (M), LpL (L) or TGRL (T). B ) (a) HAEC monolayers show changes in localization of Smad4 from cytosol to nucleus after treatment with lipolysis products compared to controls treated with M, L or T at 1 h. Treatment with lipolysis products with inhibitor ALK (10 μM) shows partial abrogation of Smad4 translocation. (b) % Accumulation of Smad4 based on counts of fluorescent nuclei. Accumulation was significantly increased after 1 h of treatment with lipolysis products (TL1h). The addition 10 μM of inhibitor ALK (TL3 + ALK) significantly reduced the observed accumulation. N = 5 coverslips/treatment group, P ≤0.05, * = TL1, TL2, TL3 compared to M, # = TL3+ALK compared to TL3 (Bar = 20 μm).

    Journal: PLoS ONE

    Article Title: TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells

    doi: 10.1371/journal.pone.0145523

    Figure Lengend Snippet: Smad2 phosphorylation at 1.5 h and Smad4 nucleus accumulation at 3 h by lipolysis products. A ) The nuclear isolated phosphorylated Smad2 was only observed with cellular exposure of lipolysis products (TL) or TGF-β1, not with Media (M), LpL (L) or TGRL (T). B ) (a) HAEC monolayers show changes in localization of Smad4 from cytosol to nucleus after treatment with lipolysis products compared to controls treated with M, L or T at 1 h. Treatment with lipolysis products with inhibitor ALK (10 μM) shows partial abrogation of Smad4 translocation. (b) % Accumulation of Smad4 based on counts of fluorescent nuclei. Accumulation was significantly increased after 1 h of treatment with lipolysis products (TL1h). The addition 10 μM of inhibitor ALK (TL3 + ALK) significantly reduced the observed accumulation. N = 5 coverslips/treatment group, P ≤0.05, * = TL1, TL2, TL3 compared to M, # = TL3+ALK compared to TL3 (Bar = 20 μm).

    Article Snippet: Human TGF-β1 (100B) and recombinant human TGF-β1 (anti TGF-β1 antibody) (MAB240) from R & D Systems Inc., Minneapolis, MN.

    Techniques: Isolation, Translocation Assay

    Increased expression of Numb in human fibrotic kidney A. Representative micrographs show the collagen deposition detected by Masson trichrome staining in human normal and fibrotic kidneys. Bar=50μm. B. Representative images of the immunohistochemical staining of Numb, TGF-β1 and p-H3 in sequential sections of renal biopsies from patients with renal tubulointerstitial fibrosis. Normal renal tissues adjacent to tumor (renal cell carcinoma) were used as normal control. Arrows indicate the co-localization of Numb, TGF-β1 and p-H3 in the injured tubules. Bar=50μm.

    Journal: Oncotarget

    Article Title: Numb contributes to renal fibrosis by promoting tubular epithelial cell cycle arrest at G2/M

    doi: 10.18632/oncotarget.8238

    Figure Lengend Snippet: Increased expression of Numb in human fibrotic kidney A. Representative micrographs show the collagen deposition detected by Masson trichrome staining in human normal and fibrotic kidneys. Bar=50μm. B. Representative images of the immunohistochemical staining of Numb, TGF-β1 and p-H3 in sequential sections of renal biopsies from patients with renal tubulointerstitial fibrosis. Normal renal tissues adjacent to tumor (renal cell carcinoma) were used as normal control. Arrows indicate the co-localization of Numb, TGF-β1 and p-H3 in the injured tubules. Bar=50μm.

    Article Snippet: After antigen repairing, sections were incubated with the primary antibody against Numb (Cell Signaling Technology, Beverly, MA, USA), p-H3(Ser10) and TGF-β1 (Abcam, Cambridge, UK), α-SMA and fibronectin (Sigma-Aldrich Sigma-Aldrich, St. Louis, MO, USA), Collagen I (Calbiochem, EMD Biosciences, Darmstadt, Germany) for 14 h at 4°C, followed by incubating with secondary antibodies (Dako, Carpinteria, CA) for 30 min.

    Techniques: Expressing, Staining, Immunohistochemistry

    Schematic model depicts the mechanism by which Numb promotes TIF In wild type mice, the expression of Numb in TECs is induced after UUO or UIRI. Increased Numb expression leads to G2/M arrest of proximal tubular cells through stabilizing p53 protein, which in turn induces the expressions of TGF-β1 and CTGF and potentiates renal fibrosis. Tubule-specific knockout Numb reduces the number of proximal tubular cells arrested at G2/M phase (empty arrow), attenuates TIF induced by UUO or UIRI.

    Journal: Oncotarget

    Article Title: Numb contributes to renal fibrosis by promoting tubular epithelial cell cycle arrest at G2/M

    doi: 10.18632/oncotarget.8238

    Figure Lengend Snippet: Schematic model depicts the mechanism by which Numb promotes TIF In wild type mice, the expression of Numb in TECs is induced after UUO or UIRI. Increased Numb expression leads to G2/M arrest of proximal tubular cells through stabilizing p53 protein, which in turn induces the expressions of TGF-β1 and CTGF and potentiates renal fibrosis. Tubule-specific knockout Numb reduces the number of proximal tubular cells arrested at G2/M phase (empty arrow), attenuates TIF induced by UUO or UIRI.

    Article Snippet: After antigen repairing, sections were incubated with the primary antibody against Numb (Cell Signaling Technology, Beverly, MA, USA), p-H3(Ser10) and TGF-β1 (Abcam, Cambridge, UK), α-SMA and fibronectin (Sigma-Aldrich Sigma-Aldrich, St. Louis, MO, USA), Collagen I (Calbiochem, EMD Biosciences, Darmstadt, Germany) for 14 h at 4°C, followed by incubating with secondary antibodies (Dako, Carpinteria, CA) for 30 min.

    Techniques: Mouse Assay, Expressing, Knock-Out

    Ectopic expression of Numb causes proximal tubular cell arrest at G2/M in vitro HK-2 cells were infected with Ad-Numb or empty vector (Ad-Ctrl). A. Cell cycle analysis by propidium iodide staining and flow cytometry in HK-2 cells. B. Bar graph depicted the percentage of cells in the different stages of the cell cycle. Symbols in the cell cycle data panels refer to the comparison of G2/M phases. C. Western blot analysis of the expression of Numb, TGF-β1, CTGF in Ad-Numb or Ad-Ctrl infected cells. GAPDH was used to verify equivalent loading. D. Graphic representation of relative abundance of TGF-β1 and CTGF to GAPDH. E. Western blot analysis of p-JNK in Ad-Numb or Ad-Ctrl infected cells (upper panel). Graphic representation of relative abundance of p-JNK to total JNK (lower panel). Data are expressed as mean±SD of three independent experiments. ** p

    Journal: Oncotarget

    Article Title: Numb contributes to renal fibrosis by promoting tubular epithelial cell cycle arrest at G2/M

    doi: 10.18632/oncotarget.8238

    Figure Lengend Snippet: Ectopic expression of Numb causes proximal tubular cell arrest at G2/M in vitro HK-2 cells were infected with Ad-Numb or empty vector (Ad-Ctrl). A. Cell cycle analysis by propidium iodide staining and flow cytometry in HK-2 cells. B. Bar graph depicted the percentage of cells in the different stages of the cell cycle. Symbols in the cell cycle data panels refer to the comparison of G2/M phases. C. Western blot analysis of the expression of Numb, TGF-β1, CTGF in Ad-Numb or Ad-Ctrl infected cells. GAPDH was used to verify equivalent loading. D. Graphic representation of relative abundance of TGF-β1 and CTGF to GAPDH. E. Western blot analysis of p-JNK in Ad-Numb or Ad-Ctrl infected cells (upper panel). Graphic representation of relative abundance of p-JNK to total JNK (lower panel). Data are expressed as mean±SD of three independent experiments. ** p

    Article Snippet: After antigen repairing, sections were incubated with the primary antibody against Numb (Cell Signaling Technology, Beverly, MA, USA), p-H3(Ser10) and TGF-β1 (Abcam, Cambridge, UK), α-SMA and fibronectin (Sigma-Aldrich Sigma-Aldrich, St. Louis, MO, USA), Collagen I (Calbiochem, EMD Biosciences, Darmstadt, Germany) for 14 h at 4°C, followed by incubating with secondary antibodies (Dako, Carpinteria, CA) for 30 min.

    Techniques: Expressing, In Vitro, Infection, Plasmid Preparation, Cell Cycle Assay, Staining, Flow Cytometry, Cytometry, Western Blot

    CPC differentiation and tube formation as a function of molecular weight of heparin and exogenous TGFβ1 loading Representative confocal microscopy images of uptake of Ac-LDL by the differentiated endothelial cells after 12 days of culture in heparin-containing HyA hydrogels (0.03Wt% heparin). Network formation was depending on both molecular weight of heparin (LMWH, UMWH, HMWH) and TGFβ1 loading in HyA hydrogel. Ac-LDL stained red, and cell nuclei counterstained blue.

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Molecular Weight and Concentration of Heparin in Hyaluronic Acid-based Matrices Modulates Growth Factor Retention Kinetics and Stem Cell Fate

    doi: 10.1016/j.jconrel.2015.04.034

    Figure Lengend Snippet: CPC differentiation and tube formation as a function of molecular weight of heparin and exogenous TGFβ1 loading Representative confocal microscopy images of uptake of Ac-LDL by the differentiated endothelial cells after 12 days of culture in heparin-containing HyA hydrogels (0.03Wt% heparin). Network formation was depending on both molecular weight of heparin (LMWH, UMWH, HMWH) and TGFβ1 loading in HyA hydrogel. Ac-LDL stained red, and cell nuclei counterstained blue.

    Article Snippet: After three DPBS washes, the amount of TGFβ1 bound to heparin-PDL-coated well was determined using sandwich ELISA kits (TGFβ1 Mouse ELISA Kit, Abcam, Cambridge, MA).

    Techniques: Molecular Weight, Confocal Microscopy, Staining

    Retention of TGFβ1 within HyA hydrogels Dependence of TGFβ1 retention kinetics on (a) heparin molecular weight (LMWH, UMWH, HMWH - 0.03 wt% heparin), and (b) weight percentage of heparin (HMWH) at various TGFβ1 concentrations (10, 20, or 40 nM).

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Molecular Weight and Concentration of Heparin in Hyaluronic Acid-based Matrices Modulates Growth Factor Retention Kinetics and Stem Cell Fate

    doi: 10.1016/j.jconrel.2015.04.034

    Figure Lengend Snippet: Retention of TGFβ1 within HyA hydrogels Dependence of TGFβ1 retention kinetics on (a) heparin molecular weight (LMWH, UMWH, HMWH - 0.03 wt% heparin), and (b) weight percentage of heparin (HMWH) at various TGFβ1 concentrations (10, 20, or 40 nM).

    Article Snippet: After three DPBS washes, the amount of TGFβ1 bound to heparin-PDL-coated well was determined using sandwich ELISA kits (TGFβ1 Mouse ELISA Kit, Abcam, Cambridge, MA).

    Techniques: Molecular Weight

    Diffusion of TGFβ1 within heparin-containing HyA hydrogels Normalized fluorescence recovery (f(t)) of FITC labeled TGFβ1 after the photobleaching are shown in top panel. Diffusion of TGFβ1 depends on the molecular weight (LMWH, UMWH, HMWH) of incorporated heparin (left, top panel) , and on the weight percentage of incorporated heparin (HMWH) (right, top panel) . Confocal microscopy images corresponding to the FRAP experiment. F initial is the time regime that corresponds to the initial fluorescence before bleaching; F 0 is the fluorescence measurement immediately after photobleaching; F t > 0 corresponds to the recovery of fluorescence after photobleaching; F ∞ corresponds to maximal recovery of fluorescence at the end of the experiment (bottom panel) .

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Molecular Weight and Concentration of Heparin in Hyaluronic Acid-based Matrices Modulates Growth Factor Retention Kinetics and Stem Cell Fate

    doi: 10.1016/j.jconrel.2015.04.034

    Figure Lengend Snippet: Diffusion of TGFβ1 within heparin-containing HyA hydrogels Normalized fluorescence recovery (f(t)) of FITC labeled TGFβ1 after the photobleaching are shown in top panel. Diffusion of TGFβ1 depends on the molecular weight (LMWH, UMWH, HMWH) of incorporated heparin (left, top panel) , and on the weight percentage of incorporated heparin (HMWH) (right, top panel) . Confocal microscopy images corresponding to the FRAP experiment. F initial is the time regime that corresponds to the initial fluorescence before bleaching; F 0 is the fluorescence measurement immediately after photobleaching; F t > 0 corresponds to the recovery of fluorescence after photobleaching; F ∞ corresponds to maximal recovery of fluorescence at the end of the experiment (bottom panel) .

    Article Snippet: After three DPBS washes, the amount of TGFβ1 bound to heparin-PDL-coated well was determined using sandwich ELISA kits (TGFβ1 Mouse ELISA Kit, Abcam, Cambridge, MA).

    Techniques: Diffusion-based Assay, Fluorescence, Labeling, Molecular Weight, Confocal Microscopy

    CPC differentiation and tube formation as a function of molecular weight of heparin and exogenous TGFβ1 loading Representative confocal microscopy images of CD31 expressed by the CPCs after 12 days of culture in heparin-containing HyA hydrogels (0.03 wt% heparin). Network formation was dependent on both molecular weight (LMWH, UMWH, HMWH) of heparin and TGFβ1 loading in HyA hydrogel. CD31 stained red, and cell nuclei counterstained blue.

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Molecular Weight and Concentration of Heparin in Hyaluronic Acid-based Matrices Modulates Growth Factor Retention Kinetics and Stem Cell Fate

    doi: 10.1016/j.jconrel.2015.04.034

    Figure Lengend Snippet: CPC differentiation and tube formation as a function of molecular weight of heparin and exogenous TGFβ1 loading Representative confocal microscopy images of CD31 expressed by the CPCs after 12 days of culture in heparin-containing HyA hydrogels (0.03 wt% heparin). Network formation was dependent on both molecular weight (LMWH, UMWH, HMWH) of heparin and TGFβ1 loading in HyA hydrogel. CD31 stained red, and cell nuclei counterstained blue.

    Article Snippet: After three DPBS washes, the amount of TGFβ1 bound to heparin-PDL-coated well was determined using sandwich ELISA kits (TGFβ1 Mouse ELISA Kit, Abcam, Cambridge, MA).

    Techniques: Molecular Weight, Confocal Microscopy, Staining

    CPCs differentiate into endothelial cells within the hydrogels The percentage of differentiated endothelial cells expressing CD31 and VE-cadherin was quantitatively measured by flow cytometry as a function of molecular weight (LMWH, UMWH, HMWH) of incorporated heparin (0.03 wt%) (left) , and on the weight percentage of incorporated heparin (HMWH) (right) at 40nM TGFβ1.

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Molecular Weight and Concentration of Heparin in Hyaluronic Acid-based Matrices Modulates Growth Factor Retention Kinetics and Stem Cell Fate

    doi: 10.1016/j.jconrel.2015.04.034

    Figure Lengend Snippet: CPCs differentiate into endothelial cells within the hydrogels The percentage of differentiated endothelial cells expressing CD31 and VE-cadherin was quantitatively measured by flow cytometry as a function of molecular weight (LMWH, UMWH, HMWH) of incorporated heparin (0.03 wt%) (left) , and on the weight percentage of incorporated heparin (HMWH) (right) at 40nM TGFβ1.

    Article Snippet: After three DPBS washes, the amount of TGFβ1 bound to heparin-PDL-coated well was determined using sandwich ELISA kits (TGFβ1 Mouse ELISA Kit, Abcam, Cambridge, MA).

    Techniques: Expressing, Flow Cytometry, Cytometry, Molecular Weight

    Emodin inhibited the Gr1 hi monocyte associated profibrogenic cytokines . (a) Immunohistochemical staining of GRN and TGF- β 1 in the liver tissues. (b) Hepatic mRNA expression of GRN and TGF- β 1 in the liver tissues. All data are expressed as the mean ± SEM. ∗∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Emodin Alleviates Liver Fibrosis of Mice by Reducing Infiltration of Gr1hi Monocytes

    doi: 10.1155/2018/5738101

    Figure Lengend Snippet: Emodin inhibited the Gr1 hi monocyte associated profibrogenic cytokines . (a) Immunohistochemical staining of GRN and TGF- β 1 in the liver tissues. (b) Hepatic mRNA expression of GRN and TGF- β 1 in the liver tissues. All data are expressed as the mean ± SEM. ∗∗ P

    Article Snippet: Then, sample sections were incubated with various primary antibody: α -smooth muscle actin (α -SMA, ab5694, Abcam, USA), collagen-I (ab34710, Abcam, USA), F4/80 (ab111101, Abcam, USA), CD45 (ab10558, Abcam, USA), CD11b (ab13357, Abcam, USA), TGF-β 1 (ab92486, Abcam, USA), GRN (ab191211, Abcam, USA), MCP-1 (ab25124, Abcam, USA), or CCL7 (orb315556, Biorbyt, UK) overnight at 4°C.

    Techniques: Immunohistochemistry, Staining, Expressing

    rLRRFIP-1 treatment affects TGF-β levels and early collagen deposition in wounds in vivo. ( A , B ) Representative images and graphical analysis of TGF-β1 and TGF-β3 levels (white fluorescence) in day 3 and day 7 wounds treated with rLRRFIP-1 (1 µg/mL) or PBS control illustrating decreased TGF-β1 and increased TGF-β3 levels in wounds in vivo following rLRRFIP-1 treatment. Epidermis is delineated by white dotted lines, e denotes epidermis, and w denotes wound dermis. Magnification ×20. Scale Bar = 50 µm; ( C , D ) Representative images and graphical analysis of total collagen levels in day 7 wounds in vivo post-treatment with rLRRFIP-1 (1 µg/mL) or PBS control illustrating increased total collagen deposition in wounds (green) following treatment with rLRRFIP-1 (1 µg/mL). Magnification ×10. Scale Bar = 100 µm. Results represent mean ± SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Recombinant Leucine-Rich Repeat Flightless-Interacting Protein-1 Improves Healing of Acute Wounds through Its Effects on Proliferation Inflammation and Collagen Deposition

    doi: 10.3390/ijms19072014

    Figure Lengend Snippet: rLRRFIP-1 treatment affects TGF-β levels and early collagen deposition in wounds in vivo. ( A , B ) Representative images and graphical analysis of TGF-β1 and TGF-β3 levels (white fluorescence) in day 3 and day 7 wounds treated with rLRRFIP-1 (1 µg/mL) or PBS control illustrating decreased TGF-β1 and increased TGF-β3 levels in wounds in vivo following rLRRFIP-1 treatment. Epidermis is delineated by white dotted lines, e denotes epidermis, and w denotes wound dermis. Magnification ×20. Scale Bar = 50 µm; ( C , D ) Representative images and graphical analysis of total collagen levels in day 7 wounds in vivo post-treatment with rLRRFIP-1 (1 µg/mL) or PBS control illustrating increased total collagen deposition in wounds (green) following treatment with rLRRFIP-1 (1 µg/mL). Magnification ×10. Scale Bar = 100 µm. Results represent mean ± SEM. * p

    Article Snippet: Immunohistochemistry protocols utilized the following antibodies: LRRFIP-1 rabbit polyclonal (Bioss Antibodies BS-12439R, Woburn, MA, USA) (1 µg/mL), proliferating cell nuclear antigen (PCNA) (#sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) (2 µg/mL), flightless I (Flii) (#sc-21716, Santa Cruz Biotechnology) (1 µg/mL), vimentin (V9) (#sc-6260, Santa Cruz Biotechnology) (1 µg/mL), keratin-10 Ab-2 (#DE-K10, Thermo Fisher Scientific, Scoresby, NSW, Australia) (1 µg/mL), CD31 rabbit polyclonal (#ab28364, Abcam, Cambridge, UK) (1 µg/mL), TLR4/CD284 (#IMG-5794, Imgenex, CA, USA) (1 µg/mL), NIMP-R14 (#sc-59338, Santa Cruz Biotechnology) (2 µg/mL), F4/80 rat monoclonal (#ab6640, Abcam) (1 µg/mL), TGF-β1 (#sc-146-G, Santa Cruz Biotechnology) (1 µg/mL), TGF-β3 (#sc-166861, Santa Cruz Biotechnology) (1 µg/mL), and species-specific secondary antibodies Alexa Flour (#A11008, Invitrogen, Melbourne, VIC, Australia) (2 µg/mL) and Alexa Flour (#A11020, Invitrogen) (2 µg/mL) following established protocols [ , ].

    Techniques: In Vivo, Fluorescence

    Effects of klotho deficiency on p-Smad2, Smad2, TGFβ1, and LC3 in diabetic kidneys. KL +/− mutant and WT male mice were injected with STZ or citrate buffer. Animals were killed 5 weeks after the initial injections. A, Representative blots

    Journal: Endocrinology

    Article Title: Genetic Deficiency of Anti-Aging Gene Klotho Exacerbates Early Nephropathy in STZ-Induced Diabetes in Male Mice

    doi: 10.1210/en.2013-1053

    Figure Lengend Snippet: Effects of klotho deficiency on p-Smad2, Smad2, TGFβ1, and LC3 in diabetic kidneys. KL +/− mutant and WT male mice were injected with STZ or citrate buffer. Animals were killed 5 weeks after the initial injections. A, Representative blots

    Article Snippet: Biological), or TGFβ1 (Santa Cruz Biotechnology; TGFβ1 and its precursor).

    Techniques: Mutagenesis, Mouse Assay, Injection

    Changes of IgA CSR in PP and LP following intestinal I/R. ( A ) Changes of TGF ‐β1 and AID in PP and LP at 5 min. and 2 hrs after reperfusion. Analysis of relative mRNA expression of these two biomarkers was shown, respectively. And representative blots were presented on the top. ( B ) Changes of GLT α and α CT s in PP and LP at 5 min. and 2 hrs after reperfusion. ( C ) Changes of IgA + B220 + and IgM + B220 + cells from PP and LP at 2 hrs after reperfusion. Representative flow cytometric profiles were presented in the left panel. Analysis of the percentage of IgA + and IgM + B220 + cells was shown in the right. Data are expressed as mean ± S.E., n = 5 or 6. Results were compared by anova with Tukey post‐test. $ P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: TGF‐β1 improves mucosal IgA dysfunction and dysbiosis following intestinal ischaemia–reperfusion in mice

    doi: 10.1111/jcmm.12789

    Figure Lengend Snippet: Changes of IgA CSR in PP and LP following intestinal I/R. ( A ) Changes of TGF ‐β1 and AID in PP and LP at 5 min. and 2 hrs after reperfusion. Analysis of relative mRNA expression of these two biomarkers was shown, respectively. And representative blots were presented on the top. ( B ) Changes of GLT α and α CT s in PP and LP at 5 min. and 2 hrs after reperfusion. ( C ) Changes of IgA + B220 + and IgM + B220 + cells from PP and LP at 2 hrs after reperfusion. Representative flow cytometric profiles were presented in the left panel. Analysis of the percentage of IgA + and IgM + B220 + cells was shown in the right. Data are expressed as mean ± S.E., n = 5 or 6. Results were compared by anova with Tukey post‐test. $ P

    Article Snippet: Transforming growth factor‐β1 group (TGF): 1 μg (100 μl) recombinant human TGF‐β1 (PeproTech Inc, Rocky Hill, NJ, USA) was infused through caudal vein at 15 min. before ischaemia.

    Techniques: Expressing, Flow Cytometry

    The experimental protocol of groups and drugs. Sham group: adjuvants were given and SMA was isolated without occlusion; injury group: adjuvants were given and SMA was occluded for 60 min.; TGF group: 1 μg TGF ‐β1 was dissolved in adjuvant 2 and then injected at 15 min. before ischaemia; SB group: 0.5 mg SB ‐431542 was dissolved in adjuvant 1 and then administrated at 30 min. before ischaemia; TGF + SB group: the same dose of TGF ‐β1 and SB ‐431542 was administrated at corresponding time‐point, respectively. Adjuvant 1 = 10% DMSO 100 μl; adjuvant 2 = 100 μl mixture (10 mM citric acid 10 μl and PBS containing 5% trehalose 90 μl); SMA : superior mesenteric artery; DMSO : dimethyl sulfoxide; TGF : transforming growth factor; SB : SB ‐431542, specific inhibitor of SMAD 2/3 transcription.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: TGF‐β1 improves mucosal IgA dysfunction and dysbiosis following intestinal ischaemia–reperfusion in mice

    doi: 10.1111/jcmm.12789

    Figure Lengend Snippet: The experimental protocol of groups and drugs. Sham group: adjuvants were given and SMA was isolated without occlusion; injury group: adjuvants were given and SMA was occluded for 60 min.; TGF group: 1 μg TGF ‐β1 was dissolved in adjuvant 2 and then injected at 15 min. before ischaemia; SB group: 0.5 mg SB ‐431542 was dissolved in adjuvant 1 and then administrated at 30 min. before ischaemia; TGF + SB group: the same dose of TGF ‐β1 and SB ‐431542 was administrated at corresponding time‐point, respectively. Adjuvant 1 = 10% DMSO 100 μl; adjuvant 2 = 100 μl mixture (10 mM citric acid 10 μl and PBS containing 5% trehalose 90 μl); SMA : superior mesenteric artery; DMSO : dimethyl sulfoxide; TGF : transforming growth factor; SB : SB ‐431542, specific inhibitor of SMAD 2/3 transcription.

    Article Snippet: Transforming growth factor‐β1 group (TGF): 1 μg (100 μl) recombinant human TGF‐β1 (PeproTech Inc, Rocky Hill, NJ, USA) was infused through caudal vein at 15 min. before ischaemia.

    Techniques: Isolation, Injection

    DIM suppressed HSC activation by inhibiting the TGF-β signalling cascade. HSC-T6 cells were co-cultured with 10 mg·mL −1 DIM for 24 h and then stimulated with 5 ng·mL −1 TGF-β1 for 24 h. (A) α-SMA mRNA levels in HSC-T6 cells were determined by qRT-PCR. (B) COL1A1 mRNA levels in HSC-T6 cells were determined by qRT-PCR. (C) α-SMA protein levels in HSC-T6 cells were measured by Western blotting. (D) COL1A1 protein levels in HSC-T6 cells were measured by Western blotting. (E) p-Smad2/3 and Smad2/3 protein levels in HSC-T6 cells were measured by Western blotting. (F) Smad7 protein levels in HSC-T6 cells were measured by Western blotting. Western blot was repeated twice with similar trends. Western blot results were semi-quantitatively analysed by Image Pro-Plus 6.0 software. Data are expressed as means ± SD of three independent experiments and each experiment contained at least three samples; * P

    Journal: British Journal of Pharmacology

    Article Title: 3,3′-Diindolylmethane ameliorates experimental hepatic fibrosis via inhibiting miR-21 expression

    doi: 10.1111/bph.12323

    Figure Lengend Snippet: DIM suppressed HSC activation by inhibiting the TGF-β signalling cascade. HSC-T6 cells were co-cultured with 10 mg·mL −1 DIM for 24 h and then stimulated with 5 ng·mL −1 TGF-β1 for 24 h. (A) α-SMA mRNA levels in HSC-T6 cells were determined by qRT-PCR. (B) COL1A1 mRNA levels in HSC-T6 cells were determined by qRT-PCR. (C) α-SMA protein levels in HSC-T6 cells were measured by Western blotting. (D) COL1A1 protein levels in HSC-T6 cells were measured by Western blotting. (E) p-Smad2/3 and Smad2/3 protein levels in HSC-T6 cells were measured by Western blotting. (F) Smad7 protein levels in HSC-T6 cells were measured by Western blotting. Western blot was repeated twice with similar trends. Western blot results were semi-quantitatively analysed by Image Pro-Plus 6.0 software. Data are expressed as means ± SD of three independent experiments and each experiment contained at least three samples; * P

    Article Snippet: Human recombinant TGF-β1 was purchased from PeproTech (Rocky Hill, NJ, USA).

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

    DIM down-regulated miR-21 expression by inhibiting AP-1 activity. HSC-T6 cells were treated with 10 mg·mL −1 DIM for 24 h and then stimulated with 5 ng·mL −1 TGF-β1 as indicated. (A) AP-1 responsive plasmid luciferase activity was measured in HSC-T6 cells after DIM treatment. (B) c-Jun protein levels in HSC-T6 cells were measured by Western blotting. (C) Fra-1 protein levels in HSC-T6 cells were measured by Western blotting. A representative example of three independent Western blot results is presented. Western blot results were semi-quantitatively analysed by Image Pro-Plus 6.0 software. Data are expressed as means ± SD of three independent experiments and each experiment contained at least three samples; * P

    Journal: British Journal of Pharmacology

    Article Title: 3,3′-Diindolylmethane ameliorates experimental hepatic fibrosis via inhibiting miR-21 expression

    doi: 10.1111/bph.12323

    Figure Lengend Snippet: DIM down-regulated miR-21 expression by inhibiting AP-1 activity. HSC-T6 cells were treated with 10 mg·mL −1 DIM for 24 h and then stimulated with 5 ng·mL −1 TGF-β1 as indicated. (A) AP-1 responsive plasmid luciferase activity was measured in HSC-T6 cells after DIM treatment. (B) c-Jun protein levels in HSC-T6 cells were measured by Western blotting. (C) Fra-1 protein levels in HSC-T6 cells were measured by Western blotting. A representative example of three independent Western blot results is presented. Western blot results were semi-quantitatively analysed by Image Pro-Plus 6.0 software. Data are expressed as means ± SD of three independent experiments and each experiment contained at least three samples; * P

    Article Snippet: Human recombinant TGF-β1 was purchased from PeproTech (Rocky Hill, NJ, USA).

    Techniques: Expressing, Activity Assay, Plasmid Preparation, Luciferase, Western Blot, Software

    DIM inhibited TGF-β signalling pathway by down-regulating the miR-21 expression. HSC-T6 cells were co-cultured with 10 mg·mL −1 DIM or transfected with 50 nM antagomir-21/antago-ctrl, and cells were further stimulated with 5 ng·mL −1 TGF-β1 for 24 h. (A) miR-21 levels in HSC-T6 cells after DIM treatment were determined by qRT-PCR. (B) α-SMA and (C) COL1A1 protein levels in HSC-T6 cells were measured by Western blotting. (D) p-Smad2/3 and Smad2/3 protein levels in HSC-T6 cells were measured by Western blotting. (E) Smad7 protein levels in HSC-T6 cells were measured by Western blotting. A representative example of three independent Western blot results is presented. Western blot results were semi-quantitatively analysed by Image Pro-Plus 6.0 software. Data are expressed as means ± SD of three independent experiments and each experiment contained at least three samples; * P

    Journal: British Journal of Pharmacology

    Article Title: 3,3′-Diindolylmethane ameliorates experimental hepatic fibrosis via inhibiting miR-21 expression

    doi: 10.1111/bph.12323

    Figure Lengend Snippet: DIM inhibited TGF-β signalling pathway by down-regulating the miR-21 expression. HSC-T6 cells were co-cultured with 10 mg·mL −1 DIM or transfected with 50 nM antagomir-21/antago-ctrl, and cells were further stimulated with 5 ng·mL −1 TGF-β1 for 24 h. (A) miR-21 levels in HSC-T6 cells after DIM treatment were determined by qRT-PCR. (B) α-SMA and (C) COL1A1 protein levels in HSC-T6 cells were measured by Western blotting. (D) p-Smad2/3 and Smad2/3 protein levels in HSC-T6 cells were measured by Western blotting. (E) Smad7 protein levels in HSC-T6 cells were measured by Western blotting. A representative example of three independent Western blot results is presented. Western blot results were semi-quantitatively analysed by Image Pro-Plus 6.0 software. Data are expressed as means ± SD of three independent experiments and each experiment contained at least three samples; * P

    Article Snippet: Human recombinant TGF-β1 was purchased from PeproTech (Rocky Hill, NJ, USA).

    Techniques: Expressing, Cell Culture, Transfection, Quantitative RT-PCR, Western Blot, Software

    Knockdown of endogenous Giα2 and RGS2 overexpression negatively regulate migration and invasion of PC3 cells in response to diverse stimuli A . Western blot analysis of Giα2 in PC3 cells lysates to validate the knockdown of endogenous Giα2 protein using siRNA. β-actin was used as an internal control. B . Cell migration of PC3 cells after transfection with control siRNA or Giα2 siRNA in response to SDF-1α (100 nM), TGFβ1 (5 ng/ml) or EGF (10 ng/ml). Results are expressed as migration index defined as the average number of cells per field for the ligand tested/the average number of cells per field for the vehicle control. Each bar represents mean ± SEM (n=3). Significant differences (P

    Journal: Journal of cellular physiology

    Article Title: Novel role of Giα2 in cell migration: Downstream of PI3-kinase/AKT and Rac1 in prostate cancer cells

    doi: 10.1002/jcp.26894

    Figure Lengend Snippet: Knockdown of endogenous Giα2 and RGS2 overexpression negatively regulate migration and invasion of PC3 cells in response to diverse stimuli A . Western blot analysis of Giα2 in PC3 cells lysates to validate the knockdown of endogenous Giα2 protein using siRNA. β-actin was used as an internal control. B . Cell migration of PC3 cells after transfection with control siRNA or Giα2 siRNA in response to SDF-1α (100 nM), TGFβ1 (5 ng/ml) or EGF (10 ng/ml). Results are expressed as migration index defined as the average number of cells per field for the ligand tested/the average number of cells per field for the vehicle control. Each bar represents mean ± SEM (n=3). Significant differences (P

    Article Snippet: Recombinant human TGFβ1 and recombinant human SDF-1α (CXCL12) were purchased from PeproTech (Rocky Hill, NJ).

    Techniques: Over Expression, Migration, Western Blot, Transfection

    Knockout, knockdown and overexpression of Giα2 have differential effects on migration and invasion in various prostate cancer cell lines A . Western blot analysis of Giα2 in lysates from PC3 cells and PC3-Giα2-null cell lines after knockout of Giα2, using CRISPR/Cas9 gene editing. α-tubulin was used as an internal control. B . Cell migration in PC3 and PC3-Giα2-null cells in response to TGFβ1 (5 ng/ml) or EGF (10 ng/ml). Results are expressed as migration index. Each bar represents mean ± SEM (n=3). Significant differences (P

    Journal: Journal of cellular physiology

    Article Title: Novel role of Giα2 in cell migration: Downstream of PI3-kinase/AKT and Rac1 in prostate cancer cells

    doi: 10.1002/jcp.26894

    Figure Lengend Snippet: Knockout, knockdown and overexpression of Giα2 have differential effects on migration and invasion in various prostate cancer cell lines A . Western blot analysis of Giα2 in lysates from PC3 cells and PC3-Giα2-null cell lines after knockout of Giα2, using CRISPR/Cas9 gene editing. α-tubulin was used as an internal control. B . Cell migration in PC3 and PC3-Giα2-null cells in response to TGFβ1 (5 ng/ml) or EGF (10 ng/ml). Results are expressed as migration index. Each bar represents mean ± SEM (n=3). Significant differences (P

    Article Snippet: Recombinant human TGFβ1 and recombinant human SDF-1α (CXCL12) were purchased from PeproTech (Rocky Hill, NJ).

    Techniques: Knock-Out, Over Expression, Migration, Western Blot, CRISPR

    PTX pretreatment and inhibition of Gβγ subunit have differential effects on PI3-kinase activation and migration in response to different ligands A . Western blot analysis of p-AKT in total cell lysates from PC3 cells after pretreatment with PTX for 1 hour, followed by OXT (100 nmol/L), TGFβ1 (5 ng/ml), or EGF (10 ng/ml) treatment for 30 minutes. Total AKT served as a loading and normalization control. B . Quantitative differences in levels of p-AKT in different groups. Data are expressed as mean ± SEM (n=3) and analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups in a given category (P

    Journal: Journal of cellular physiology

    Article Title: Novel role of Giα2 in cell migration: Downstream of PI3-kinase/AKT and Rac1 in prostate cancer cells

    doi: 10.1002/jcp.26894

    Figure Lengend Snippet: PTX pretreatment and inhibition of Gβγ subunit have differential effects on PI3-kinase activation and migration in response to different ligands A . Western blot analysis of p-AKT in total cell lysates from PC3 cells after pretreatment with PTX for 1 hour, followed by OXT (100 nmol/L), TGFβ1 (5 ng/ml), or EGF (10 ng/ml) treatment for 30 minutes. Total AKT served as a loading and normalization control. B . Quantitative differences in levels of p-AKT in different groups. Data are expressed as mean ± SEM (n=3) and analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups in a given category (P

    Article Snippet: Recombinant human TGFβ1 and recombinant human SDF-1α (CXCL12) were purchased from PeproTech (Rocky Hill, NJ).

    Techniques: Inhibition, Activation Assay, Migration, Western Blot, Modification

    Knockdown of endogenous Giα2 has differential effects on PI3-kinase activation and cell migration in response to diverse ligands A . Western blot analysis of p-AKT and p-EGFR in PC3 cells lysates, transfected with control siRNA or Giα2 siRNA in response to OXT (100 nmol/L), TGFβ1 (5 ng/ml) or EGF (3 ng/ml). Total AKT and EGFR served as loading and normalization controls. B . Quantitative analysis of p-AKT activation in PC3 cells. Data is presented as mean ± SEM (n=3) and analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups in a given category (P

    Journal: Journal of cellular physiology

    Article Title: Novel role of Giα2 in cell migration: Downstream of PI3-kinase/AKT and Rac1 in prostate cancer cells

    doi: 10.1002/jcp.26894

    Figure Lengend Snippet: Knockdown of endogenous Giα2 has differential effects on PI3-kinase activation and cell migration in response to diverse ligands A . Western blot analysis of p-AKT and p-EGFR in PC3 cells lysates, transfected with control siRNA or Giα2 siRNA in response to OXT (100 nmol/L), TGFβ1 (5 ng/ml) or EGF (3 ng/ml). Total AKT and EGFR served as loading and normalization controls. B . Quantitative analysis of p-AKT activation in PC3 cells. Data is presented as mean ± SEM (n=3) and analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups in a given category (P

    Article Snippet: Recombinant human TGFβ1 and recombinant human SDF-1α (CXCL12) were purchased from PeproTech (Rocky Hill, NJ).

    Techniques: Activation Assay, Migration, Western Blot, Transfection, Modification

    (a) 2.5 × 10 5 Naïve CD4 + CD62L + T-cells were isolated and stimulated for 72 hrs with or without anti-CD3 (1 µg/ml) in the presence of IL-6 (40 ng/ml)/TGF-β1 (2 ng/ml) and tested for IL-17 and IFN-γ, (b) IL-17 was measured in cultures of T-cells stimulated as above that were grown in the presence of IFN-γ (20 ng/ml), (c) CCR6 and IL-23R RNA was measured in cells as cultured in (a). (d) 2.5 × 10 5 WC1 + γδ T-cells were isolated and cultured with IL-6/TGF-β1, as above, with or without anti-CD3 and IL-17 was measured after 72 hrs. (e) γδ T-cells were cultured as in (d) with added IFN-γ (20 ng/ml) before measurement of IL-17. (f) CCR6 and IL-23R RNA was measured in γδ T-cells from (d) above. Data are means of triplicates ± SD and representative of one of four animals tested. Data was analysed using one way ANOVA (*P =

    Journal: Scientific Reports

    Article Title: Two distinct populations of Bovine IL-17+ T-cells can be induced and WC1+IL-17+γδ T-cells are effective killers of protozoan parasites

    doi: 10.1038/srep05431

    Figure Lengend Snippet: (a) 2.5 × 10 5 Naïve CD4 + CD62L + T-cells were isolated and stimulated for 72 hrs with or without anti-CD3 (1 µg/ml) in the presence of IL-6 (40 ng/ml)/TGF-β1 (2 ng/ml) and tested for IL-17 and IFN-γ, (b) IL-17 was measured in cultures of T-cells stimulated as above that were grown in the presence of IFN-γ (20 ng/ml), (c) CCR6 and IL-23R RNA was measured in cells as cultured in (a). (d) 2.5 × 10 5 WC1 + γδ T-cells were isolated and cultured with IL-6/TGF-β1, as above, with or without anti-CD3 and IL-17 was measured after 72 hrs. (e) γδ T-cells were cultured as in (d) with added IFN-γ (20 ng/ml) before measurement of IL-17. (f) CCR6 and IL-23R RNA was measured in γδ T-cells from (d) above. Data are means of triplicates ± SD and representative of one of four animals tested. Data was analysed using one way ANOVA (*P =

    Article Snippet: ELISAs & Recombinant cytokines Recombinant proteins used were as follows, bovine IL-6, bovine IFN-γ (Kingfisher Biotech) and human TGF-β1 (Peprotech).

    Techniques: Isolation, Cell Culture

    Hs578T cells have mesenchymal features, express high ZEB1 levels and are sensitized to TGFβ signaling. (a) Expression of ZEB1 and ZEB2 mRNA in different subtypes of breast cancer cells (basal‐A, red, basal‐B, gray, and Luminal, blue) based on expression values derived from the GOBO database. Red boxes indicate the cell lines with considerably high ZEB1 mRNA expression, which were selected for further analysis. (b) Immunoblot showing expression of ZEB1 in Hs578T, MDA‐MB‐231, and T47D cells, after stimulation with or without TGFβ1 for 24 hr. β‐Actin serves as protein loading control and pSmad2 as a marker of TGFβ pathway activation; “*” indicates an unspecific protein band. (c) Immunoblot showing expression of epithelial and mesenchymal proteins in Hs578T cells in response to TGFβ1 stimulation for the indicated time periods in the absence (DMSO) or presence of GW6604 (5 µM) inhibitor. Cntrl indicates no stimulation with TGFβ1. β‐Actin serves as protein loading control and pSmad2 as a marker of TGFβ pathway activation. (d) Quantification of binding of ZEB1 to the CDH1 promoter by ChIP–qPCR. An isotype–specific immunoglobulin control precipitation serves as reference. Statistical significance ( p ‐value

    Journal: Journal of Cellular Physiology

    Article Title: Genome–wide binding of transcription factor ZEB1 in triple‐negative breast cancer cells. Genome–wide binding of transcription factor ZEB1 in triple‐negative breast cancer cells

    doi: 10.1002/jcp.26634

    Figure Lengend Snippet: Hs578T cells have mesenchymal features, express high ZEB1 levels and are sensitized to TGFβ signaling. (a) Expression of ZEB1 and ZEB2 mRNA in different subtypes of breast cancer cells (basal‐A, red, basal‐B, gray, and Luminal, blue) based on expression values derived from the GOBO database. Red boxes indicate the cell lines with considerably high ZEB1 mRNA expression, which were selected for further analysis. (b) Immunoblot showing expression of ZEB1 in Hs578T, MDA‐MB‐231, and T47D cells, after stimulation with or without TGFβ1 for 24 hr. β‐Actin serves as protein loading control and pSmad2 as a marker of TGFβ pathway activation; “*” indicates an unspecific protein band. (c) Immunoblot showing expression of epithelial and mesenchymal proteins in Hs578T cells in response to TGFβ1 stimulation for the indicated time periods in the absence (DMSO) or presence of GW6604 (5 µM) inhibitor. Cntrl indicates no stimulation with TGFβ1. β‐Actin serves as protein loading control and pSmad2 as a marker of TGFβ pathway activation. (d) Quantification of binding of ZEB1 to the CDH1 promoter by ChIP–qPCR. An isotype–specific immunoglobulin control precipitation serves as reference. Statistical significance ( p ‐value

    Article Snippet: Cells starved for 18 hr in serum‐free DMEM or RPMI were stimulated with 5 ng/ml TGFβ1 (recombinant human TGFβ1, PeproTech Nordic, Stockholm, Sweden).

    Techniques: Expressing, Derivative Assay, Multiple Displacement Amplification, Marker, Activation Assay, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    TGF-β1–mediated migration is reduced in Itga3 –/– keratinocytes.

    Journal:

    Article Title: ?3?1 integrin-controlled Smad7 regulates reepithelialization during wound healing in mice

    doi: 10.1172/JCI33538

    Figure Lengend Snippet: TGF-β1–mediated migration is reduced in Itga3 –/– keratinocytes.

    Article Snippet: Without changing the medium, each well was treated with either 1 ng/ml TGF-β1 (Peprotech) or left untreated.

    Techniques: Migration

    Enrichment of miR-744 reduced phosphorylation of Smad3 and increased the production of SARA in epithelial cells from normal or severe asthma subject. ( A ) Western blot analysis was conducted to detected total and phosphorylated Smad3 or SARA expression in NBECs and SABECs with TGF-β1 overexpression vector or (and) miR-744 mimic or miR-NC transfection. ( B–D ) Relative densitometry analyses of proteins were conducted using Image Lab software. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-744 Inhibits Proliferation of Bronchial Epithelial Cells by Regulating Smad3 Pathway via Targeting Transforming Growth Factor-β1 (TGF-β1) in Severe Asthma

    doi: 10.12659/MSM.912412

    Figure Lengend Snippet: Enrichment of miR-744 reduced phosphorylation of Smad3 and increased the production of SARA in epithelial cells from normal or severe asthma subject. ( A ) Western blot analysis was conducted to detected total and phosphorylated Smad3 or SARA expression in NBECs and SABECs with TGF-β1 overexpression vector or (and) miR-744 mimic or miR-NC transfection. ( B–D ) Relative densitometry analyses of proteins were conducted using Image Lab software. * P

    Article Snippet: The membranes were blocked with 5% non-fat milk for 2 h at room temperature and then incubated overnight at 4o C with primary monoclonal antibodies against proliferating cell nuclear antigen (PCNA), TGF-β1, phosphorylated mothers against decapentaplegic homolog3 (p-Smad3), Smad3, and Smad anchor for receptor activation (SARA) or β-actin (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation, Transfection, Software

    miR-744 directly targeted the 3′-UTR of TGF-β1. ( A ) Putative targeting sequences of miR-744 and 3′-UTR of TGF-β1 were provided by TargetScan. ( B, C ) Luciferase assays were used to probe the interaction between miR-744 and TGF-β1 in BEAS-2B cells with miR-744 mimic or inhibitor transfection. ( D–F ) Effects of miR-744 on TGF-β1 mRNA and protein expressions were evaluated in bronchial epithelial cells. ( G ) Interaction between miR-744 and TGF-β1 was analyzed by Ago2 RIP. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-744 Inhibits Proliferation of Bronchial Epithelial Cells by Regulating Smad3 Pathway via Targeting Transforming Growth Factor-β1 (TGF-β1) in Severe Asthma

    doi: 10.12659/MSM.912412

    Figure Lengend Snippet: miR-744 directly targeted the 3′-UTR of TGF-β1. ( A ) Putative targeting sequences of miR-744 and 3′-UTR of TGF-β1 were provided by TargetScan. ( B, C ) Luciferase assays were used to probe the interaction between miR-744 and TGF-β1 in BEAS-2B cells with miR-744 mimic or inhibitor transfection. ( D–F ) Effects of miR-744 on TGF-β1 mRNA and protein expressions were evaluated in bronchial epithelial cells. ( G ) Interaction between miR-744 and TGF-β1 was analyzed by Ago2 RIP. * P

    Article Snippet: The membranes were blocked with 5% non-fat milk for 2 h at room temperature and then incubated overnight at 4o C with primary monoclonal antibodies against proliferating cell nuclear antigen (PCNA), TGF-β1, phosphorylated mothers against decapentaplegic homolog3 (p-Smad3), Smad3, and Smad anchor for receptor activation (SARA) or β-actin (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Luciferase, Transfection

    TGF-β1 was required for miR-744-mediated cell proliferation in bronchial epithelial cells from normal or severe asthma subjects. ( A, B ) Cell proliferation was measured in NBEC and SABEC cells transfected with TGF-β1 overexpression vector or (and) miR-744 mimic or miR-NC. ( C, D ) The abundances of PCNA were detected in transfected NBEC and SABEC cells. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-744 Inhibits Proliferation of Bronchial Epithelial Cells by Regulating Smad3 Pathway via Targeting Transforming Growth Factor-β1 (TGF-β1) in Severe Asthma

    doi: 10.12659/MSM.912412

    Figure Lengend Snippet: TGF-β1 was required for miR-744-mediated cell proliferation in bronchial epithelial cells from normal or severe asthma subjects. ( A, B ) Cell proliferation was measured in NBEC and SABEC cells transfected with TGF-β1 overexpression vector or (and) miR-744 mimic or miR-NC. ( C, D ) The abundances of PCNA were detected in transfected NBEC and SABEC cells. * P

    Article Snippet: The membranes were blocked with 5% non-fat milk for 2 h at room temperature and then incubated overnight at 4o C with primary monoclonal antibodies against proliferating cell nuclear antigen (PCNA), TGF-β1, phosphorylated mothers against decapentaplegic homolog3 (p-Smad3), Smad3, and Smad anchor for receptor activation (SARA) or β-actin (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection, Over Expression, Plasmid Preparation

    TNF-α suppresses TGFβ1-dependent production of SMαA protein and mRNA in human pulmonary myofibroblasts. (a) hPFBs were pretreated with TNF-α (10 ng/ml) for 2 h followed by exposure to TGFβ1 (5 ng/ml) for another

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor ?1-mediated Activation of the Smooth Muscle ?-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-? via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

    doi: 10.1091/mbc.E08-10-0994

    Figure Lengend Snippet: TNF-α suppresses TGFβ1-dependent production of SMαA protein and mRNA in human pulmonary myofibroblasts. (a) hPFBs were pretreated with TNF-α (10 ng/ml) for 2 h followed by exposure to TGFβ1 (5 ng/ml) for another

    Article Snippet: Recombinant human TGFβ1 and human TNF-α (R & D Systems, Minneapolis, MN) were added to cell cultures for varying periods and doses as noted in the text.

    Techniques:

    Effects of exogenous Mek1 signaling on SMαA promoter activity in TGFβ1-treated human pulmonary myofibroblasts. hPFBs were transfected with the SMαA promoter: CAT reporter fusion gene (VSMP4) together with expression plasmids encoding

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor ?1-mediated Activation of the Smooth Muscle ?-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-? via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

    doi: 10.1091/mbc.E08-10-0994

    Figure Lengend Snippet: Effects of exogenous Mek1 signaling on SMαA promoter activity in TGFβ1-treated human pulmonary myofibroblasts. hPFBs were transfected with the SMαA promoter: CAT reporter fusion gene (VSMP4) together with expression plasmids encoding

    Article Snippet: Recombinant human TGFβ1 and human TNF-α (R & D Systems, Minneapolis, MN) were added to cell cultures for varying periods and doses as noted in the text.

    Techniques: Activity Assay, Transfection, Expressing

    Intact Mek/Erk signaling was required for TNF-α to repress activation of the SMαA gene by TGFβ1 in human pulmonary myofibroblasts. As shown in a, Quiescent hPFBs were pretreated with DMSO (vehicle), SP600125 (10 μM, JNK

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor ?1-mediated Activation of the Smooth Muscle ?-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-? via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

    doi: 10.1091/mbc.E08-10-0994

    Figure Lengend Snippet: Intact Mek/Erk signaling was required for TNF-α to repress activation of the SMαA gene by TGFβ1 in human pulmonary myofibroblasts. As shown in a, Quiescent hPFBs were pretreated with DMSO (vehicle), SP600125 (10 μM, JNK

    Article Snippet: Recombinant human TGFβ1 and human TNF-α (R & D Systems, Minneapolis, MN) were added to cell cultures for varying periods and doses as noted in the text.

    Techniques: Activation Assay

    Egr-1 overexpression abrogates TGFβ1- and Smad-activated SMαA promoter activity in human pulmonary myofibroblasts. hPFBs were transiently cotransfected with the VSMP4 reporter gene plus various amounts (0.1–1 μg) of expression

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor ?1-mediated Activation of the Smooth Muscle ?-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-? via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

    doi: 10.1091/mbc.E08-10-0994

    Figure Lengend Snippet: Egr-1 overexpression abrogates TGFβ1- and Smad-activated SMαA promoter activity in human pulmonary myofibroblasts. hPFBs were transiently cotransfected with the VSMP4 reporter gene plus various amounts (0.1–1 μg) of expression

    Article Snippet: Recombinant human TGFβ1 and human TNF-α (R & D Systems, Minneapolis, MN) were added to cell cultures for varying periods and doses as noted in the text.

    Techniques: Over Expression, Activity Assay, Expressing

    Egr-1 expression in human pulmonary fibroblasts and interaction with a TGFβ1-response element in the SMαA promoter. (a) Transiently elevated Egr-1 protein expression in nuclear preparations from TNF-α–treated hPFBs. Quiescent

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor ?1-mediated Activation of the Smooth Muscle ?-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-? via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

    doi: 10.1091/mbc.E08-10-0994

    Figure Lengend Snippet: Egr-1 expression in human pulmonary fibroblasts and interaction with a TGFβ1-response element in the SMαA promoter. (a) Transiently elevated Egr-1 protein expression in nuclear preparations from TNF-α–treated hPFBs. Quiescent

    Article Snippet: Recombinant human TGFβ1 and human TNF-α (R & D Systems, Minneapolis, MN) were added to cell cultures for varying periods and doses as noted in the text.

    Techniques: Expressing

    HSP72 blocks p-Smad3 nuclear translocation and accumulation in a time- and dose-dependent manner. (A) NRK-52E cells were treated with 10 ng/ml of TGF-β1 for 30 minutes. Representative confocal microscopic images showed the cellular localization of Smad3 (red) and HSP72 (green) by indirect immunofluorescence staining in cells. Original magnification × 400. (B) The cellular localization of p-Smad3 was examined by confocal microscopic images of p-Smad3 (red), HSP72 (green), and nuclear staining (blue) in empty-vector control and HSP72 overexpressing cells after 10 ng/ml of TGF-β1 exposure for 24 hours. Original magnification × 400. (C) NRK-52E cells were treated with wild-type HSP72 (30 and 50 nM) or HSP72 siRNA after 10 ng/ml of TGF-β1 exposure for 30 minutes. Empty vector served as negative control. Smad3 and p-Smad3 nuclear translocation and accumulation were assessed by Western blot analysis. (D) Wild-type HSP72 or HSP72 siRNA were individually expressed in NRK-52E cells before treatment with 10 ng/ml of TGF-β1 for different time periods before the assessment of p-Smad3 nuclear translocation and accumulation. Empty vector served as negative control. Smad3 and p-Smad3 nuclear translocation and accumulation were assessed by Western blot analysis. (E, F) Quantitative determination of the relative abundance of p-Smad3 nuclear accumulation among different groups. Data are expressed as mean ± SEM of three experiments. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: HSP72 Inhibits Smad3 Activation and Nuclear Translocation in Renal Epithelial-to-Mesenchymal Transition

    doi: 10.1681/ASN.2009050552

    Figure Lengend Snippet: HSP72 blocks p-Smad3 nuclear translocation and accumulation in a time- and dose-dependent manner. (A) NRK-52E cells were treated with 10 ng/ml of TGF-β1 for 30 minutes. Representative confocal microscopic images showed the cellular localization of Smad3 (red) and HSP72 (green) by indirect immunofluorescence staining in cells. Original magnification × 400. (B) The cellular localization of p-Smad3 was examined by confocal microscopic images of p-Smad3 (red), HSP72 (green), and nuclear staining (blue) in empty-vector control and HSP72 overexpressing cells after 10 ng/ml of TGF-β1 exposure for 24 hours. Original magnification × 400. (C) NRK-52E cells were treated with wild-type HSP72 (30 and 50 nM) or HSP72 siRNA after 10 ng/ml of TGF-β1 exposure for 30 minutes. Empty vector served as negative control. Smad3 and p-Smad3 nuclear translocation and accumulation were assessed by Western blot analysis. (D) Wild-type HSP72 or HSP72 siRNA were individually expressed in NRK-52E cells before treatment with 10 ng/ml of TGF-β1 for different time periods before the assessment of p-Smad3 nuclear translocation and accumulation. Empty vector served as negative control. Smad3 and p-Smad3 nuclear translocation and accumulation were assessed by Western blot analysis. (E, F) Quantitative determination of the relative abundance of p-Smad3 nuclear accumulation among different groups. Data are expressed as mean ± SEM of three experiments. * P

    Article Snippet: Reagents were purchased from the following vendors: human recombinant TGF-β1 was from R & D Systems (Minneapolis, MN); anti-phospho-Smad3 (p-Smad3, Ser423/425), anti-phospho-Smad2 (p-Smad2, Ser465/467), anti-Smad2, anti-Smad3, and anti-Smad7 from Cell Signaling Technology (Beverly, MA); anti-E-cadherin antibody and anti-HA tag antibody from BD (Biosciences Pharmingen, San Jose, CA); anti-α-SMA was from DAKO (Cupertino, CA); anti-HSP72 from Stressgen Biotechnologies (Victoria, British Columbia, Canada); and anti-fibrillarin from Abcam (Cambridge, MA); anti-β-actin from Boster (Wuhan, China).

    Techniques: Translocation Assay, Immunofluorescence, Staining, Plasmid Preparation, Negative Control, Western Blot

    PBD is required for HSP72 to prevent Smad3 activation. (A) NRK-52E cells were transiently transfected with plasmids encoding Wt-HSP72, HSP72-ΔPBD, or HSP72-ΔNLS followed by incubation with 10 ng/ml of TGF-β1 for 30 minutes. Smad protein levels were examined by Western blotting. (B, C) p-Smad3 and Smad7 contents were quantitatively analyzed using a densitometer. Values are mean ± SEM; n = 3 per treatment. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: HSP72 Inhibits Smad3 Activation and Nuclear Translocation in Renal Epithelial-to-Mesenchymal Transition

    doi: 10.1681/ASN.2009050552

    Figure Lengend Snippet: PBD is required for HSP72 to prevent Smad3 activation. (A) NRK-52E cells were transiently transfected with plasmids encoding Wt-HSP72, HSP72-ΔPBD, or HSP72-ΔNLS followed by incubation with 10 ng/ml of TGF-β1 for 30 minutes. Smad protein levels were examined by Western blotting. (B, C) p-Smad3 and Smad7 contents were quantitatively analyzed using a densitometer. Values are mean ± SEM; n = 3 per treatment. * P

    Article Snippet: Reagents were purchased from the following vendors: human recombinant TGF-β1 was from R & D Systems (Minneapolis, MN); anti-phospho-Smad3 (p-Smad3, Ser423/425), anti-phospho-Smad2 (p-Smad2, Ser465/467), anti-Smad2, anti-Smad3, and anti-Smad7 from Cell Signaling Technology (Beverly, MA); anti-E-cadherin antibody and anti-HA tag antibody from BD (Biosciences Pharmingen, San Jose, CA); anti-α-SMA was from DAKO (Cupertino, CA); anti-HSP72 from Stressgen Biotechnologies (Victoria, British Columbia, Canada); and anti-fibrillarin from Abcam (Cambridge, MA); anti-β-actin from Boster (Wuhan, China).

    Techniques: Activation Assay, Transfection, Incubation, Western Blot

    HSP72 suppresses activation of the TGF-β/Smads pathway. (A) Serum-deprived NRK-52E cells were treated with 10 ng/ml of TGF-β1 for the indicated time period. Cell lysates were probed with antibodies against p-Smad3, Smad3, p-Smad2, Smad2, Smad7 or β-actin. (B) NRK-52E cells were transfected with pcDNA3.1-HA-Wt-HSP72 (30 and 50 nM) or specific HSP72 siRNA were stimulated with 10 ng/ml of TGF-β1 for 30 minutes. (C through E) Smad protein content evaluated by Western blotting with densitometric analysis of the effect of HSP72 expression on p-Smad3, p-Smad2 and Smad7 content normalized with Smad2, Smad3, or β-actin content in TGF-β1-treated cells. Data are expressed as mean ± SEM; n = 3 per treatment; * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: HSP72 Inhibits Smad3 Activation and Nuclear Translocation in Renal Epithelial-to-Mesenchymal Transition

    doi: 10.1681/ASN.2009050552

    Figure Lengend Snippet: HSP72 suppresses activation of the TGF-β/Smads pathway. (A) Serum-deprived NRK-52E cells were treated with 10 ng/ml of TGF-β1 for the indicated time period. Cell lysates were probed with antibodies against p-Smad3, Smad3, p-Smad2, Smad2, Smad7 or β-actin. (B) NRK-52E cells were transfected with pcDNA3.1-HA-Wt-HSP72 (30 and 50 nM) or specific HSP72 siRNA were stimulated with 10 ng/ml of TGF-β1 for 30 minutes. (C through E) Smad protein content evaluated by Western blotting with densitometric analysis of the effect of HSP72 expression on p-Smad3, p-Smad2 and Smad7 content normalized with Smad2, Smad3, or β-actin content in TGF-β1-treated cells. Data are expressed as mean ± SEM; n = 3 per treatment; * P

    Article Snippet: Reagents were purchased from the following vendors: human recombinant TGF-β1 was from R & D Systems (Minneapolis, MN); anti-phospho-Smad3 (p-Smad3, Ser423/425), anti-phospho-Smad2 (p-Smad2, Ser465/467), anti-Smad2, anti-Smad3, and anti-Smad7 from Cell Signaling Technology (Beverly, MA); anti-E-cadherin antibody and anti-HA tag antibody from BD (Biosciences Pharmingen, San Jose, CA); anti-α-SMA was from DAKO (Cupertino, CA); anti-HSP72 from Stressgen Biotechnologies (Victoria, British Columbia, Canada); and anti-fibrillarin from Abcam (Cambridge, MA); anti-β-actin from Boster (Wuhan, China).

    Techniques: Activation Assay, Transfection, Western Blot, Expressing

    HSP72 abrogates EMT. (A) Wild-type or HSP72 mutants were overexpressed in NRK-52E cells before treatment with 10 ng/ml of TGF-β1 for 48 hours. Cell lysates were analyzed by immunoblotting with antibodies against E-cadherin, α-SMA, or HSP72. β-actin served as a loading control. (B, C) Densitometric analysis of the effect of HSP72 on E-cadherin and α-SMA expression. Expression of these proteins was normalized to β-actin content in cells treated as described in panel A. Data are expressed as mean ± SEM; n = 3 per treatment; * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: HSP72 Inhibits Smad3 Activation and Nuclear Translocation in Renal Epithelial-to-Mesenchymal Transition

    doi: 10.1681/ASN.2009050552

    Figure Lengend Snippet: HSP72 abrogates EMT. (A) Wild-type or HSP72 mutants were overexpressed in NRK-52E cells before treatment with 10 ng/ml of TGF-β1 for 48 hours. Cell lysates were analyzed by immunoblotting with antibodies against E-cadherin, α-SMA, or HSP72. β-actin served as a loading control. (B, C) Densitometric analysis of the effect of HSP72 on E-cadherin and α-SMA expression. Expression of these proteins was normalized to β-actin content in cells treated as described in panel A. Data are expressed as mean ± SEM; n = 3 per treatment; * P

    Article Snippet: Reagents were purchased from the following vendors: human recombinant TGF-β1 was from R & D Systems (Minneapolis, MN); anti-phospho-Smad3 (p-Smad3, Ser423/425), anti-phospho-Smad2 (p-Smad2, Ser465/467), anti-Smad2, anti-Smad3, and anti-Smad7 from Cell Signaling Technology (Beverly, MA); anti-E-cadherin antibody and anti-HA tag antibody from BD (Biosciences Pharmingen, San Jose, CA); anti-α-SMA was from DAKO (Cupertino, CA); anti-HSP72 from Stressgen Biotechnologies (Victoria, British Columbia, Canada); and anti-fibrillarin from Abcam (Cambridge, MA); anti-β-actin from Boster (Wuhan, China).

    Techniques: Expressing

    Effect of TGF-β1 on Smad3 deficient versus WT mast cell proliferation

    Journal: Journal of pediatric gastroenterology and nutrition

    Article Title: Smad3 deficient mice have reduced esophageal fibrosis and angiogenesis in a mouse model of egg induced eosinophilic esophagitis

    doi: 10.1097/MPG.0000000000000343

    Figure Lengend Snippet: Effect of TGF-β1 on Smad3 deficient versus WT mast cell proliferation

    Article Snippet: In brief, total RNA was extracted with RNA-STAT-60 (Tel-Test) and reverse transcribed with Oligo-dT and SuperScript II kit (Life Technologies). qPCR was performed with TaqMan PCR Master Mix and TGF-β1, primers (Applied Biosystems).

    Techniques:

    Overexpression of circPTK2 enhances TIF1γ expression and inhibits TGF-β-induced EMT and invasion of NSCLC cells in vitro. a TIF1γ mRNA and protein levels in A549 and H226 cells transiently overexpressing circPTK2. Relative TIF1γ expression was determined with normalization against β-actin. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing circPTK2 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 0.5 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. c A549 and H226 cells transiently overexpressing circPTK2 were serum-starved for 24 h and then treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Snail and N-cadherin protein levels were determined by western blot. β-actin was used as internal control. d , e A549 and H226 cells transiently overexpressing circPTK2 were treated as above and subjected to the transwell migration and invasion assays. Migrated and invasive cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. * P

    Journal: Molecular Cancer

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer

    doi: 10.1186/s12943-018-0889-7

    Figure Lengend Snippet: Overexpression of circPTK2 enhances TIF1γ expression and inhibits TGF-β-induced EMT and invasion of NSCLC cells in vitro. a TIF1γ mRNA and protein levels in A549 and H226 cells transiently overexpressing circPTK2. Relative TIF1γ expression was determined with normalization against β-actin. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing circPTK2 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 0.5 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. c A549 and H226 cells transiently overexpressing circPTK2 were serum-starved for 24 h and then treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Snail and N-cadherin protein levels were determined by western blot. β-actin was used as internal control. d , e A549 and H226 cells transiently overexpressing circPTK2 were treated as above and subjected to the transwell migration and invasion assays. Migrated and invasive cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. * P

    Article Snippet: Briefly, A549 and H226 cells transiently overexpressing miR-429/miR-200b-3p or circPTK2 were incubated with TGF-β1 (5 ng/ml) in Transwell plates (BD Biosciences) for 24 h and 48 h. Then cells were allowed to migrate through an 8-μM pored membrane or invade through Matrigel-coated membrane.

    Techniques: Over Expression, Expressing, In Vitro, Quantitative RT-PCR, Western Blot, Migration, Staining

    CircPTK2 overexpression attenuates NSCLC cell metastasis in vivo, and circPTK2 levels were lower in metastatic NSCLC tissues than non-metastatic counterparts. a CircPTK2 expression in A549 cells stably overexpressing circPTK2. A549 stable cell line overexpressing circPTK2 was generated as described in Methods. pLCDH-circPTK2-copGFP(T2A)Puro lentiviral expression vector ( upper ) was used to stably overexpress circPTK2. The empty vector was served as negative control. CircPTK2 expression was determined by qRT-PCR ( bottom left ). CircPTK2 expression in circPTK2-overexpressed A549 cells was determined using northern blots. RNase R was used to digest linear RNA ( bottom right ). b Schematic flowchart of the in vivo metastasis experiments with A549 cells stably transfected with pLCDH-circPTK2 or vector (i.v.) and TGF-β1 (i.p.) injected into BALB/c nude mice ( n = 6 mice per group in circPTK2 + TGF-β1 and vector + TGF-β1). c Representative images showing metastatic nodules established in lung taken from the mice injected with circPTK2-overexpressed A549 cells or vector control cells ( upper ). Scale bar, 4 mm. Haematoxylin and eosin (H E) staining was performed for histological confirmation of metastasizing tumor cells in lung ( bottom ). Scale bar, 100 μm. d Gross view of metastatic nodules developed in liver ( upper left ) and dot plots showing the number of metastatic nodules in liver ( upper right , n = 6 mice per group). Scale bar, 4 mm. Microscopic images of H E staining for liver metastases ( bottom left ) and the distribution of the number of metastases in per section of liver ( bottom right , n = 6 mice per group). Scale bar, 100 μm. Yellow and green arrowheads indicate metastatic nodules and micrometastases. e Representative images indicating metastatic nodules developed in pericardium ( upper , n = 6 mice per group) and H E staining of heart ( bottom ). Scale bar, 1 mm or 100 μm. f , g CircPTK2 and TIF1γ mRNA expression levels in human lung epithelial and NSCLC cells. β-actin was used as internal control. Each qRT-PCR analysis was performed in triplicate. h , i qRT-PCR analysis of circPTK2 and TIF1γ mRNA levels in 73 human NSCLC tissues and paired noncancerous lung tissues. Mean values are indicted by solid bars, and values are expressed as mean ± SEM. T, NSCLC tissues; N, paired noncancerous lung tissues. j , k Relative expression of circPTK2 and TIF1γ mRNA in 73 paired NSCLC tissues. Y -axis represents the log 10 transformed fold change of T/N expression ratios of circPTK2 and TIF1γ mRNA. The number of each specimen is shown below x -axis. l Correlation between circPTK2 level and TIF1γ mRNA expression in 73 paired NSCLC tissues. X and y axes represent the T/N expression ratios of circPTK2 and TIF1γ mRNA, respectively. m Relative expression (T/N) of circPTK2 in metastatic ( n = 41) and non-metastatic ( n = 32) NSCLC tissues. Metastatic tissues were from NSCLC patients with lymph node metastasis or distant metastasis and non-metastatic tissues were from NSCLC patients without any metastasis, respectively. * P

    Journal: Molecular Cancer

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer

    doi: 10.1186/s12943-018-0889-7

    Figure Lengend Snippet: CircPTK2 overexpression attenuates NSCLC cell metastasis in vivo, and circPTK2 levels were lower in metastatic NSCLC tissues than non-metastatic counterparts. a CircPTK2 expression in A549 cells stably overexpressing circPTK2. A549 stable cell line overexpressing circPTK2 was generated as described in Methods. pLCDH-circPTK2-copGFP(T2A)Puro lentiviral expression vector ( upper ) was used to stably overexpress circPTK2. The empty vector was served as negative control. CircPTK2 expression was determined by qRT-PCR ( bottom left ). CircPTK2 expression in circPTK2-overexpressed A549 cells was determined using northern blots. RNase R was used to digest linear RNA ( bottom right ). b Schematic flowchart of the in vivo metastasis experiments with A549 cells stably transfected with pLCDH-circPTK2 or vector (i.v.) and TGF-β1 (i.p.) injected into BALB/c nude mice ( n = 6 mice per group in circPTK2 + TGF-β1 and vector + TGF-β1). c Representative images showing metastatic nodules established in lung taken from the mice injected with circPTK2-overexpressed A549 cells or vector control cells ( upper ). Scale bar, 4 mm. Haematoxylin and eosin (H E) staining was performed for histological confirmation of metastasizing tumor cells in lung ( bottom ). Scale bar, 100 μm. d Gross view of metastatic nodules developed in liver ( upper left ) and dot plots showing the number of metastatic nodules in liver ( upper right , n = 6 mice per group). Scale bar, 4 mm. Microscopic images of H E staining for liver metastases ( bottom left ) and the distribution of the number of metastases in per section of liver ( bottom right , n = 6 mice per group). Scale bar, 100 μm. Yellow and green arrowheads indicate metastatic nodules and micrometastases. e Representative images indicating metastatic nodules developed in pericardium ( upper , n = 6 mice per group) and H E staining of heart ( bottom ). Scale bar, 1 mm or 100 μm. f , g CircPTK2 and TIF1γ mRNA expression levels in human lung epithelial and NSCLC cells. β-actin was used as internal control. Each qRT-PCR analysis was performed in triplicate. h , i qRT-PCR analysis of circPTK2 and TIF1γ mRNA levels in 73 human NSCLC tissues and paired noncancerous lung tissues. Mean values are indicted by solid bars, and values are expressed as mean ± SEM. T, NSCLC tissues; N, paired noncancerous lung tissues. j , k Relative expression of circPTK2 and TIF1γ mRNA in 73 paired NSCLC tissues. Y -axis represents the log 10 transformed fold change of T/N expression ratios of circPTK2 and TIF1γ mRNA. The number of each specimen is shown below x -axis. l Correlation between circPTK2 level and TIF1γ mRNA expression in 73 paired NSCLC tissues. X and y axes represent the T/N expression ratios of circPTK2 and TIF1γ mRNA, respectively. m Relative expression (T/N) of circPTK2 in metastatic ( n = 41) and non-metastatic ( n = 32) NSCLC tissues. Metastatic tissues were from NSCLC patients with lymph node metastasis or distant metastasis and non-metastatic tissues were from NSCLC patients without any metastasis, respectively. * P

    Article Snippet: Briefly, A549 and H226 cells transiently overexpressing miR-429/miR-200b-3p or circPTK2 were incubated with TGF-β1 (5 ng/ml) in Transwell plates (BD Biosciences) for 24 h and 48 h. Then cells were allowed to migrate through an 8-μM pored membrane or invade through Matrigel-coated membrane.

    Techniques: Over Expression, In Vivo, Expressing, Stable Transfection, Generated, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Northern Blot, Transfection, Injection, Mouse Assay, Staining, Transformation Assay

    TIF1γ and circPTK2 are down-regulated during TGF-β-induced EMT in NSCLC cells. a A549 cells underwent epithelial-mesenchymal transition (EMT) after TGF-β1 (5 ng/ml) treatment for 24 h. Cell morphology was observed and photographed with a phase-contrast microscope ( upper ). Scale bar, 50 μm. The expression of EMT-related makers including E-cadherin, N-cadherin and Vimentin ( bottom left ), and TIF1γ protein ( bottom right ) were examined by western blot. β-actin was used as internal control. Densitometry values for each protein were normalized to β-actin and shown below the corresponding bands. b RNA from epithelial and mesenchymal A549 cells were subjected to Arraystar Human circRNA Array analysis as described in Methods. Hierarchical cluster analysis (heat map) of microarray data was used to show the significant expression of circRNAs when comparing mesenchymal cells with epithelial cells ( left ). Red and green denoted high and low expression, respectively. Each column represents a test sample and each row represents a circRNA. Each group (treated with TGF-β1 for 0 h or 24 h) was analyzed in triplicate. In a zoomed-in view of partial ( right ), the expression of circPTK2 (hsa_circRNA_104703) was indicated as an arrow. c The sketch of genomic locus of circPTK2 in PTK2 gene. The expression of circPTK2 (circBase ID: hsa_circ_0008305) was validated by RT-PCR followed by sanger sequencing. Red arrows represent divergent primers, which are used to amplify the genome region of circPTK2 containing the back-splice junction site (JCT). d In A549 or H226 cells, divergent primers amplify circPTK2 JCT in cDNA but not in genomic DNA (gDNA), convergent primers amplify both circPTK2 JCT and linear PTK2 Exon 9. GAPDH was used as linear control. Red and black arrows represent divergent and convergent primers, respectively. Divergent primers spanning circPTK2 JCT yield a product of 110 bp, while the convergent primers amplifying PTK2 exon 9 yield a product of 141 bp. e Endogenous circPTK2 expression in A549 cells was validated by northern blots. RNase R was used to digest linear RNA. f Representative image of RNA fluorescence in situ hybridization for endogenous circPTK2 in A549 cells. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. g qRT-PCR analysis of circPTK2 expression in A549 and H226 cells treated with TGF-β1 for 24 h. Relative circPTK2 expression was determined with normalization against β-actin. h, i qRT-PCR analysis of miR-429/miR-200b-3p expression levels in A549 and H226 cells treated with TGF-β1 for 24 h. U6 was used as internal control. * P

    Journal: Molecular Cancer

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer

    doi: 10.1186/s12943-018-0889-7

    Figure Lengend Snippet: TIF1γ and circPTK2 are down-regulated during TGF-β-induced EMT in NSCLC cells. a A549 cells underwent epithelial-mesenchymal transition (EMT) after TGF-β1 (5 ng/ml) treatment for 24 h. Cell morphology was observed and photographed with a phase-contrast microscope ( upper ). Scale bar, 50 μm. The expression of EMT-related makers including E-cadherin, N-cadherin and Vimentin ( bottom left ), and TIF1γ protein ( bottom right ) were examined by western blot. β-actin was used as internal control. Densitometry values for each protein were normalized to β-actin and shown below the corresponding bands. b RNA from epithelial and mesenchymal A549 cells were subjected to Arraystar Human circRNA Array analysis as described in Methods. Hierarchical cluster analysis (heat map) of microarray data was used to show the significant expression of circRNAs when comparing mesenchymal cells with epithelial cells ( left ). Red and green denoted high and low expression, respectively. Each column represents a test sample and each row represents a circRNA. Each group (treated with TGF-β1 for 0 h or 24 h) was analyzed in triplicate. In a zoomed-in view of partial ( right ), the expression of circPTK2 (hsa_circRNA_104703) was indicated as an arrow. c The sketch of genomic locus of circPTK2 in PTK2 gene. The expression of circPTK2 (circBase ID: hsa_circ_0008305) was validated by RT-PCR followed by sanger sequencing. Red arrows represent divergent primers, which are used to amplify the genome region of circPTK2 containing the back-splice junction site (JCT). d In A549 or H226 cells, divergent primers amplify circPTK2 JCT in cDNA but not in genomic DNA (gDNA), convergent primers amplify both circPTK2 JCT and linear PTK2 Exon 9. GAPDH was used as linear control. Red and black arrows represent divergent and convergent primers, respectively. Divergent primers spanning circPTK2 JCT yield a product of 110 bp, while the convergent primers amplifying PTK2 exon 9 yield a product of 141 bp. e Endogenous circPTK2 expression in A549 cells was validated by northern blots. RNase R was used to digest linear RNA. f Representative image of RNA fluorescence in situ hybridization for endogenous circPTK2 in A549 cells. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. g qRT-PCR analysis of circPTK2 expression in A549 and H226 cells treated with TGF-β1 for 24 h. Relative circPTK2 expression was determined with normalization against β-actin. h, i qRT-PCR analysis of miR-429/miR-200b-3p expression levels in A549 and H226 cells treated with TGF-β1 for 24 h. U6 was used as internal control. * P

    Article Snippet: Briefly, A549 and H226 cells transiently overexpressing miR-429/miR-200b-3p or circPTK2 were incubated with TGF-β1 (5 ng/ml) in Transwell plates (BD Biosciences) for 24 h and 48 h. Then cells were allowed to migrate through an 8-μM pored membrane or invade through Matrigel-coated membrane.

    Techniques: Microscopy, Expressing, Western Blot, Microarray, Reverse Transcription Polymerase Chain Reaction, Sequencing, Northern Blot, Fluorescence, In Situ Hybridization, Quantitative RT-PCR

    miR-429 enhances TGF-β-induced EMT and invasion in NSCLC cells. a After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing miR-429 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 2 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing miR-429 were treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Western blot analysis was performed to examine the expression of N-cadherin, which was normalized to β-actin. c A549 and H226 cells transiently overexpressing miR-429 were treated as above and allowed to migrate through an 8-μM pore in transwells. Migrated cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. d Cells were treated as above and allowed to invade through Matrigel-coated membrane in transwells. Invasive cells were stained and counted under a light microscope. Scale bar, 100 μm. e After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing anti-miR-429 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 2 h, respectively. qRT-PCR analysis was done to determine the relative Snail mRNA expression. f After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing anti-miR-429 were treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. N-cadherin expression was analyzed by western blot. g A549 and H226 cells transiently overexpressing anti-miR-429 were treated as above and allowed to migrate through an 8-μM pore in transwells. Migrated cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. h Cells were treated as above and allowed to invade through Matrigel-coated membrane in transwells. Invasive cells were stained and counted under a light microscope. Scale bar, 100 μm. ** P

    Journal: Molecular Cancer

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer

    doi: 10.1186/s12943-018-0889-7

    Figure Lengend Snippet: miR-429 enhances TGF-β-induced EMT and invasion in NSCLC cells. a After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing miR-429 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 2 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing miR-429 were treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Western blot analysis was performed to examine the expression of N-cadherin, which was normalized to β-actin. c A549 and H226 cells transiently overexpressing miR-429 were treated as above and allowed to migrate through an 8-μM pore in transwells. Migrated cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. d Cells were treated as above and allowed to invade through Matrigel-coated membrane in transwells. Invasive cells were stained and counted under a light microscope. Scale bar, 100 μm. e After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing anti-miR-429 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 2 h, respectively. qRT-PCR analysis was done to determine the relative Snail mRNA expression. f After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing anti-miR-429 were treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. N-cadherin expression was analyzed by western blot. g A549 and H226 cells transiently overexpressing anti-miR-429 were treated as above and allowed to migrate through an 8-μM pore in transwells. Migrated cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. h Cells were treated as above and allowed to invade through Matrigel-coated membrane in transwells. Invasive cells were stained and counted under a light microscope. Scale bar, 100 μm. ** P

    Article Snippet: Briefly, A549 and H226 cells transiently overexpressing miR-429/miR-200b-3p or circPTK2 were incubated with TGF-β1 (5 ng/ml) in Transwell plates (BD Biosciences) for 24 h and 48 h. Then cells were allowed to migrate through an 8-μM pored membrane or invade through Matrigel-coated membrane.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Light Microscopy

    Co-culture of macrophages and myofibroblasts induces α-SMA, TGF-β1 and Smad3 expression. ( A ) Representative immunohistochemical staining of α-SMA with 3-D co-culture in the present of Ang II. Scale bars: 50 µm. ( B ) Real-time PCR quantification of α-SMA and TGF-β1 mRNA expression. ( C ) Immunofluorescence staining of protein level of phosphorylated Smad3 (p-Smad3; green) and DDR2 (red) or Immunofluorescence staining of TGF-β1 (green) and DDR2 (red) in co-cultured WT macrophages and fibroblasts with neutralizing antibody to IL-6 and co-cultured IL-6-/- macrophages and fibroblasts with recombinant IL-6 (rIL-6) treatment. Scale bars: 50 µm. (n = 6 per group). ( D ) Western blot analysis and quantification of protein level of TGF-β1 and p-Smad3 in 3-D co-culture (n = 4 per group). * P

    Journal: PLoS ONE

    Article Title: Macrophage-Stimulated Cardiac Fibroblast Production of IL-6 Is Essential for TGF ?/Smad Activation and Cardiac Fibrosis Induced by Angiotensin II

    doi: 10.1371/journal.pone.0035144

    Figure Lengend Snippet: Co-culture of macrophages and myofibroblasts induces α-SMA, TGF-β1 and Smad3 expression. ( A ) Representative immunohistochemical staining of α-SMA with 3-D co-culture in the present of Ang II. Scale bars: 50 µm. ( B ) Real-time PCR quantification of α-SMA and TGF-β1 mRNA expression. ( C ) Immunofluorescence staining of protein level of phosphorylated Smad3 (p-Smad3; green) and DDR2 (red) or Immunofluorescence staining of TGF-β1 (green) and DDR2 (red) in co-cultured WT macrophages and fibroblasts with neutralizing antibody to IL-6 and co-cultured IL-6-/- macrophages and fibroblasts with recombinant IL-6 (rIL-6) treatment. Scale bars: 50 µm. (n = 6 per group). ( D ) Western blot analysis and quantification of protein level of TGF-β1 and p-Smad3 in 3-D co-culture (n = 4 per group). * P

    Article Snippet: Then the nitrocellulose membrane was incubated with the primary antibodies anti-GAPDH (1∶5000 diluted in TBS-T, Kangwei, China), anti-α-SMA (1∶1000 dilution, Abcam), anti-p-Smad3 or anti-TGF-β1 (both 1∶1000 dilution, Santa Cruz Biotechnology), then with IR Dye-conjugated secondary antibodies (1∶5000, Rockland Immunochemicals, Gilbertsville, PA) for 1 h. Images were quantified by use of the Odyssey infrared imaging system (LI-COR Biosciences Lincoln, NE).

    Techniques: Co-Culture Assay, Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Immunofluorescence, Cell Culture, Recombinant, Western Blot

    IL-6 promotes Ang II-induced cardiac fibrosis and inflammation. ( A ) Ang II-induced hypertensive cardiac fibrosis in C57BL/6 wild-type (WT) and IL-6-/- mice (n = 6 per group). (Left) Masson’s trichrome staining (blue) of ventricular sections analyzed for fibrotic areas at day 7 after Ang II infusion (n = 6 mice/group). Scale bars: 500 µm (top), 100 µm (bottom). Fibrotic area is stained blue. (Right) Quantification of fibrotic areas. ( B ) Western blot analysis and quantification of protein levels of transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in WT and IL-6-/- hearts (n = 6 per group). Normalization is to GAPDH level. ( C ) Representative immunohistochemical staining of collagen I, TGF-β1 and α-SMA in WT and IL-6-/- hearts. Scale bars: 50 µm and quantification (right). ( D ) Immunohistochemical staining and quantification of Mac-2 in WT and IL-6-/- hearts. Scale bars: 50 µm. Data represent the mean±SEM (n = 5 per group). * P

    Journal: PLoS ONE

    Article Title: Macrophage-Stimulated Cardiac Fibroblast Production of IL-6 Is Essential for TGF ?/Smad Activation and Cardiac Fibrosis Induced by Angiotensin II

    doi: 10.1371/journal.pone.0035144

    Figure Lengend Snippet: IL-6 promotes Ang II-induced cardiac fibrosis and inflammation. ( A ) Ang II-induced hypertensive cardiac fibrosis in C57BL/6 wild-type (WT) and IL-6-/- mice (n = 6 per group). (Left) Masson’s trichrome staining (blue) of ventricular sections analyzed for fibrotic areas at day 7 after Ang II infusion (n = 6 mice/group). Scale bars: 500 µm (top), 100 µm (bottom). Fibrotic area is stained blue. (Right) Quantification of fibrotic areas. ( B ) Western blot analysis and quantification of protein levels of transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in WT and IL-6-/- hearts (n = 6 per group). Normalization is to GAPDH level. ( C ) Representative immunohistochemical staining of collagen I, TGF-β1 and α-SMA in WT and IL-6-/- hearts. Scale bars: 50 µm and quantification (right). ( D ) Immunohistochemical staining and quantification of Mac-2 in WT and IL-6-/- hearts. Scale bars: 50 µm. Data represent the mean±SEM (n = 5 per group). * P

    Article Snippet: Then the nitrocellulose membrane was incubated with the primary antibodies anti-GAPDH (1∶5000 diluted in TBS-T, Kangwei, China), anti-α-SMA (1∶1000 dilution, Abcam), anti-p-Smad3 or anti-TGF-β1 (both 1∶1000 dilution, Santa Cruz Biotechnology), then with IR Dye-conjugated secondary antibodies (1∶5000, Rockland Immunochemicals, Gilbertsville, PA) for 1 h. Images were quantified by use of the Odyssey infrared imaging system (LI-COR Biosciences Lincoln, NE).

    Techniques: Mouse Assay, Staining, Western Blot, Immunohistochemistry

    HGF decreases α-SMA expression in HUVECs through TGF-β/Smad and Akt/mTOR/p70S6K signaling pathways. ( A, B ) HUVECs treated with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours. We collected equal amounts of protein from whole cell lysates, which we analyzed by western blotting with antibodies against phosphorylated Smad 2, Smad 3, Akt, mTOR, p70 S6K, Erk1/2, c-Jun. ( C, D ) HUVECs were pretreated for one hour with MK2206 (10 μmol/L) ( C ), and SB431542 (10 μmol/L) ( D ), which are specific chemical inhibitors for Akt and Smad, respectively. Subsequently, we treated cells with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours. We collected cells one hour after HGF and/or TGF-β1 stimulation. We analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against phosphorylated and total Akt ( C ), phosphorylated and total mTOR ( C ), phosphorylated and total p70S6K ( C ), phosphorylated and total Smad 2 ( D ), phosphorylated and total Smad 3 ( D ). ( C, D ) We collected HUVECs 48 hours after HGF and/or TGF-β1 stimulation and analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against α-SMA and GAPDH.

    Journal: Annals of Transplantation

    Article Title: Antifibrotic Effects of Hepatocyte Growth Factor on Endothelial-to-Mesenchymal Transition via Transforming Growth Factor-Beta1 (TGF-β1)/Smad and Akt/mTOR/P70S6K Signaling Pathways

    doi: 10.12659/AOT.906700

    Figure Lengend Snippet: HGF decreases α-SMA expression in HUVECs through TGF-β/Smad and Akt/mTOR/p70S6K signaling pathways. ( A, B ) HUVECs treated with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours. We collected equal amounts of protein from whole cell lysates, which we analyzed by western blotting with antibodies against phosphorylated Smad 2, Smad 3, Akt, mTOR, p70 S6K, Erk1/2, c-Jun. ( C, D ) HUVECs were pretreated for one hour with MK2206 (10 μmol/L) ( C ), and SB431542 (10 μmol/L) ( D ), which are specific chemical inhibitors for Akt and Smad, respectively. Subsequently, we treated cells with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours. We collected cells one hour after HGF and/or TGF-β1 stimulation. We analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against phosphorylated and total Akt ( C ), phosphorylated and total mTOR ( C ), phosphorylated and total p70S6K ( C ), phosphorylated and total Smad 2 ( D ), phosphorylated and total Smad 3 ( D ). ( C, D ) We collected HUVECs 48 hours after HGF and/or TGF-β1 stimulation and analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against α-SMA and GAPDH.

    Article Snippet: Then, we performed western blotting by incubation with primary antibodies of anti-GAPDH (1: 200, Abcam, Cambridge, MA, USA), anti-α-SMA (1: 2,500, Abcam), anti-CD31 (1: 1,000, Abcam), anti-TGF-β1 (1: 250, Abcam), anti-HGF (1: 500, Abcam), anti-Akt (1: 1,000, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt (Ser473, 1: 1,000, Cell Signaling Technology), anti-mTOR (1: 1,000, Cell Signaling Technology,), anti-phospho-mTOR (Ser2448, 1: 1,000, Cell Signaling Technology), anti-p70S6K (1: 1,000, Cell Signaling Technology), anti-phospho-p70S6K (Thr389, 1: 1,000, Cell Signaling Technology), anti-Erk 1/2 (1: 1,000, Cell Signaling Technology) anti-phospho-Erk 1/2 (Thr202/Tyr204, 1: 1,000, Cell Signaling Technology), anti-c-Jun (1: 1,000, Cell Signaling Technology) and anti-phospho-c-Jun (Ser73, 1: 1,000, Cell Signaling Technology), anti-Smad 2 (1: 1,000, Cell Signaling Technology), anti-phospho-Smad 2 (Ser465/467, 1: 1,000, Cell Signaling Technology), anti-Smad 3 (1: 1,000, Cell Signaling Technology) and anti-phospho-Smad 3 (Ser423/425, 1: 1,000, Cell Signaling Technology), followed by incubation with anti-rabbit or anti-mouse secondary antibody (1: 1,000, Abcam).

    Techniques: Expressing, Western Blot

    HGF weakens the motility and migration ability of HUVECs and secretion of extracellular matrix. ( A–F ) HUVECs incubated with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours ( A, C, E, F ). ( A, B ) We wounded HUVECs and manually counted migrated cells. The motility index is expressed as the fold-change relative to unstimulated control cells ( B ). ( C, D ) We seeded a total of 5×10 4 HUVECs in the top chamber, and stained and quantified cells that migrated through the membrane. The migration index is expressed as the fold-change relative to unstimulated control cells ( D ). We collected the supernatant of the cultured HUVECs for ELISA to determine total concentrations of collagen-I ( E ) and fibronectin ( F ). Data are presented as mean ±SD of three independent experiments; * p

    Journal: Annals of Transplantation

    Article Title: Antifibrotic Effects of Hepatocyte Growth Factor on Endothelial-to-Mesenchymal Transition via Transforming Growth Factor-Beta1 (TGF-β1)/Smad and Akt/mTOR/P70S6K Signaling Pathways

    doi: 10.12659/AOT.906700

    Figure Lengend Snippet: HGF weakens the motility and migration ability of HUVECs and secretion of extracellular matrix. ( A–F ) HUVECs incubated with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours ( A, C, E, F ). ( A, B ) We wounded HUVECs and manually counted migrated cells. The motility index is expressed as the fold-change relative to unstimulated control cells ( B ). ( C, D ) We seeded a total of 5×10 4 HUVECs in the top chamber, and stained and quantified cells that migrated through the membrane. The migration index is expressed as the fold-change relative to unstimulated control cells ( D ). We collected the supernatant of the cultured HUVECs for ELISA to determine total concentrations of collagen-I ( E ) and fibronectin ( F ). Data are presented as mean ±SD of three independent experiments; * p

    Article Snippet: Then, we performed western blotting by incubation with primary antibodies of anti-GAPDH (1: 200, Abcam, Cambridge, MA, USA), anti-α-SMA (1: 2,500, Abcam), anti-CD31 (1: 1,000, Abcam), anti-TGF-β1 (1: 250, Abcam), anti-HGF (1: 500, Abcam), anti-Akt (1: 1,000, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt (Ser473, 1: 1,000, Cell Signaling Technology), anti-mTOR (1: 1,000, Cell Signaling Technology,), anti-phospho-mTOR (Ser2448, 1: 1,000, Cell Signaling Technology), anti-p70S6K (1: 1,000, Cell Signaling Technology), anti-phospho-p70S6K (Thr389, 1: 1,000, Cell Signaling Technology), anti-Erk 1/2 (1: 1,000, Cell Signaling Technology) anti-phospho-Erk 1/2 (Thr202/Tyr204, 1: 1,000, Cell Signaling Technology), anti-c-Jun (1: 1,000, Cell Signaling Technology) and anti-phospho-c-Jun (Ser73, 1: 1,000, Cell Signaling Technology), anti-Smad 2 (1: 1,000, Cell Signaling Technology), anti-phospho-Smad 2 (Ser465/467, 1: 1,000, Cell Signaling Technology), anti-Smad 3 (1: 1,000, Cell Signaling Technology) and anti-phospho-Smad 3 (Ser423/425, 1: 1,000, Cell Signaling Technology), followed by incubation with anti-rabbit or anti-mouse secondary antibody (1: 1,000, Abcam).

    Techniques: Migration, Incubation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

    A proposed model illustrating the antifibrotic effects of HGF on TGF-β1-induced EndMT.

    Journal: Annals of Transplantation

    Article Title: Antifibrotic Effects of Hepatocyte Growth Factor on Endothelial-to-Mesenchymal Transition via Transforming Growth Factor-Beta1 (TGF-β1)/Smad and Akt/mTOR/P70S6K Signaling Pathways

    doi: 10.12659/AOT.906700

    Figure Lengend Snippet: A proposed model illustrating the antifibrotic effects of HGF on TGF-β1-induced EndMT.

    Article Snippet: Then, we performed western blotting by incubation with primary antibodies of anti-GAPDH (1: 200, Abcam, Cambridge, MA, USA), anti-α-SMA (1: 2,500, Abcam), anti-CD31 (1: 1,000, Abcam), anti-TGF-β1 (1: 250, Abcam), anti-HGF (1: 500, Abcam), anti-Akt (1: 1,000, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt (Ser473, 1: 1,000, Cell Signaling Technology), anti-mTOR (1: 1,000, Cell Signaling Technology,), anti-phospho-mTOR (Ser2448, 1: 1,000, Cell Signaling Technology), anti-p70S6K (1: 1,000, Cell Signaling Technology), anti-phospho-p70S6K (Thr389, 1: 1,000, Cell Signaling Technology), anti-Erk 1/2 (1: 1,000, Cell Signaling Technology) anti-phospho-Erk 1/2 (Thr202/Tyr204, 1: 1,000, Cell Signaling Technology), anti-c-Jun (1: 1,000, Cell Signaling Technology) and anti-phospho-c-Jun (Ser73, 1: 1,000, Cell Signaling Technology), anti-Smad 2 (1: 1,000, Cell Signaling Technology), anti-phospho-Smad 2 (Ser465/467, 1: 1,000, Cell Signaling Technology), anti-Smad 3 (1: 1,000, Cell Signaling Technology) and anti-phospho-Smad 3 (Ser423/425, 1: 1,000, Cell Signaling Technology), followed by incubation with anti-rabbit or anti-mouse secondary antibody (1: 1,000, Abcam).

    Techniques:

    HGF weakens the motility and migration ability of HRGECs and secretion of extracellular matrix. ( A–F ) HRGECs were incubated with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours ( A, C, E, F ). ( A, B ) We wounded HRGECs and manually counted migrated cells. The motility index is expressed as the fold-change relative to unstimulated control cells ( B ). ( C, D ) We seeded a total of 5× 10 4 HRGECs in the top chamber and stained and quantified cells that migrated through the membrane. The migration index is expressed as the fold-change relative to unstimulated control cells ( D ). We collected the supernatant of the cultured HRGECs for ELISA to determine total concentrations of collagen-I ( E ) and fibronectin ( F ). Data are presented as mean ±SD of three independent experiments; * p

    Journal: Annals of Transplantation

    Article Title: Antifibrotic Effects of Hepatocyte Growth Factor on Endothelial-to-Mesenchymal Transition via Transforming Growth Factor-Beta1 (TGF-β1)/Smad and Akt/mTOR/P70S6K Signaling Pathways

    doi: 10.12659/AOT.906700

    Figure Lengend Snippet: HGF weakens the motility and migration ability of HRGECs and secretion of extracellular matrix. ( A–F ) HRGECs were incubated with 40 ng/mL HGF and/or 5 ng/mL TGF-β1 for 48 hours ( A, C, E, F ). ( A, B ) We wounded HRGECs and manually counted migrated cells. The motility index is expressed as the fold-change relative to unstimulated control cells ( B ). ( C, D ) We seeded a total of 5× 10 4 HRGECs in the top chamber and stained and quantified cells that migrated through the membrane. The migration index is expressed as the fold-change relative to unstimulated control cells ( D ). We collected the supernatant of the cultured HRGECs for ELISA to determine total concentrations of collagen-I ( E ) and fibronectin ( F ). Data are presented as mean ±SD of three independent experiments; * p

    Article Snippet: Then, we performed western blotting by incubation with primary antibodies of anti-GAPDH (1: 200, Abcam, Cambridge, MA, USA), anti-α-SMA (1: 2,500, Abcam), anti-CD31 (1: 1,000, Abcam), anti-TGF-β1 (1: 250, Abcam), anti-HGF (1: 500, Abcam), anti-Akt (1: 1,000, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt (Ser473, 1: 1,000, Cell Signaling Technology), anti-mTOR (1: 1,000, Cell Signaling Technology,), anti-phospho-mTOR (Ser2448, 1: 1,000, Cell Signaling Technology), anti-p70S6K (1: 1,000, Cell Signaling Technology), anti-phospho-p70S6K (Thr389, 1: 1,000, Cell Signaling Technology), anti-Erk 1/2 (1: 1,000, Cell Signaling Technology) anti-phospho-Erk 1/2 (Thr202/Tyr204, 1: 1,000, Cell Signaling Technology), anti-c-Jun (1: 1,000, Cell Signaling Technology) and anti-phospho-c-Jun (Ser73, 1: 1,000, Cell Signaling Technology), anti-Smad 2 (1: 1,000, Cell Signaling Technology), anti-phospho-Smad 2 (Ser465/467, 1: 1,000, Cell Signaling Technology), anti-Smad 3 (1: 1,000, Cell Signaling Technology) and anti-phospho-Smad 3 (Ser423/425, 1: 1,000, Cell Signaling Technology), followed by incubation with anti-rabbit or anti-mouse secondary antibody (1: 1,000, Abcam).

    Techniques: Migration, Incubation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

    HGF attenuates α-SMA expressions and increases CD31 expression in HUVECs and HRGECs. HUVECs ( A–D ) and HRGECs ( E–H ) were incubated with 5 ng/mL TGF-β1 and/or 20 ng/mL or 40 ng/mL HGF for 48 hours. We analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against α-SMA, CD31 and GAPDH. We isolated and reversed-transcribed total RNA, and subjected the resultant RNA to quantitative real-time PCR to detect gene expression of α-SMA and CD31. We normalized the quantitative real-time PCR results to β2-macroglobulin. These are expressed as the fold-change relative to unstimulated control cells in HUVECs ( C, D ) and HRGECs ( G, H ). The relative abundance of mRNAs are presented as mean ±SD of three independent experiments. * p

    Journal: Annals of Transplantation

    Article Title: Antifibrotic Effects of Hepatocyte Growth Factor on Endothelial-to-Mesenchymal Transition via Transforming Growth Factor-Beta1 (TGF-β1)/Smad and Akt/mTOR/P70S6K Signaling Pathways

    doi: 10.12659/AOT.906700

    Figure Lengend Snippet: HGF attenuates α-SMA expressions and increases CD31 expression in HUVECs and HRGECs. HUVECs ( A–D ) and HRGECs ( E–H ) were incubated with 5 ng/mL TGF-β1 and/or 20 ng/mL or 40 ng/mL HGF for 48 hours. We analyzed equal amounts of protein from whole cell lysates by western blotting with antibodies against α-SMA, CD31 and GAPDH. We isolated and reversed-transcribed total RNA, and subjected the resultant RNA to quantitative real-time PCR to detect gene expression of α-SMA and CD31. We normalized the quantitative real-time PCR results to β2-macroglobulin. These are expressed as the fold-change relative to unstimulated control cells in HUVECs ( C, D ) and HRGECs ( G, H ). The relative abundance of mRNAs are presented as mean ±SD of three independent experiments. * p

    Article Snippet: Then, we performed western blotting by incubation with primary antibodies of anti-GAPDH (1: 200, Abcam, Cambridge, MA, USA), anti-α-SMA (1: 2,500, Abcam), anti-CD31 (1: 1,000, Abcam), anti-TGF-β1 (1: 250, Abcam), anti-HGF (1: 500, Abcam), anti-Akt (1: 1,000, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt (Ser473, 1: 1,000, Cell Signaling Technology), anti-mTOR (1: 1,000, Cell Signaling Technology,), anti-phospho-mTOR (Ser2448, 1: 1,000, Cell Signaling Technology), anti-p70S6K (1: 1,000, Cell Signaling Technology), anti-phospho-p70S6K (Thr389, 1: 1,000, Cell Signaling Technology), anti-Erk 1/2 (1: 1,000, Cell Signaling Technology) anti-phospho-Erk 1/2 (Thr202/Tyr204, 1: 1,000, Cell Signaling Technology), anti-c-Jun (1: 1,000, Cell Signaling Technology) and anti-phospho-c-Jun (Ser73, 1: 1,000, Cell Signaling Technology), anti-Smad 2 (1: 1,000, Cell Signaling Technology), anti-phospho-Smad 2 (Ser465/467, 1: 1,000, Cell Signaling Technology), anti-Smad 3 (1: 1,000, Cell Signaling Technology) and anti-phospho-Smad 3 (Ser423/425, 1: 1,000, Cell Signaling Technology), followed by incubation with anti-rabbit or anti-mouse secondary antibody (1: 1,000, Abcam).

    Techniques: Expressing, Incubation, Western Blot, Isolation, Real-time Polymerase Chain Reaction

    The effects of actin microfilament inhibition on basal and TGF β 1-stimulated superficial zone protein (SZP) media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 1, 6, and 10 μM cytochalasin

    Journal: Tissue Engineering. Part A

    Article Title: Transforming Growth Factor β-Induced Superficial Zone Protein Accumulation in the Surface Zone of Articular Cartilage Is Dependent on the Cytoskeleton

    doi: 10.1089/ten.tea.2013.0043

    Figure Lengend Snippet: The effects of actin microfilament inhibition on basal and TGF β 1-stimulated superficial zone protein (SZP) media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 1, 6, and 10 μM cytochalasin

    Article Snippet: After the media exchange, chondrocytes were pretreated with an inhibitor for 1 h, before the addition of 3 ng/mL TGF β 1 (R & D Systems, Minneapolis, MN).

    Techniques: Inhibition

    The effects of Cdc42 and Rac1 inhibition on basal and TGF β 1-stimulated SZP media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 5 and 20 μM ML141, a reversible, noncompetitive inhibitor of

    Journal: Tissue Engineering. Part A

    Article Title: Transforming Growth Factor β-Induced Superficial Zone Protein Accumulation in the Surface Zone of Articular Cartilage Is Dependent on the Cytoskeleton

    doi: 10.1089/ten.tea.2013.0043

    Figure Lengend Snippet: The effects of Cdc42 and Rac1 inhibition on basal and TGF β 1-stimulated SZP media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 5 and 20 μM ML141, a reversible, noncompetitive inhibitor of

    Article Snippet: After the media exchange, chondrocytes were pretreated with an inhibitor for 1 h, before the addition of 3 ng/mL TGF β 1 (R & D Systems, Minneapolis, MN).

    Techniques: Inhibition

    The effects of microtubule modulation on basal and TGF β 1-stimulated SZP media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 1 and 10 μM colchicine, a microtubule polymerization inhibitor,

    Journal: Tissue Engineering. Part A

    Article Title: Transforming Growth Factor β-Induced Superficial Zone Protein Accumulation in the Surface Zone of Articular Cartilage Is Dependent on the Cytoskeleton

    doi: 10.1089/ten.tea.2013.0043

    Figure Lengend Snippet: The effects of microtubule modulation on basal and TGF β 1-stimulated SZP media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 1 and 10 μM colchicine, a microtubule polymerization inhibitor,

    Article Snippet: After the media exchange, chondrocytes were pretreated with an inhibitor for 1 h, before the addition of 3 ng/mL TGF β 1 (R & D Systems, Minneapolis, MN).

    Techniques:

    The effects of Rho Kinase (ROCK) inhibition and RhoA activation on basal and TGF β 1-stimulated SZP media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 10 and 50 μM Y27632, a competitive inhibitor

    Journal: Tissue Engineering. Part A

    Article Title: Transforming Growth Factor β-Induced Superficial Zone Protein Accumulation in the Surface Zone of Articular Cartilage Is Dependent on the Cytoskeleton

    doi: 10.1089/ten.tea.2013.0043

    Figure Lengend Snippet: The effects of Rho Kinase (ROCK) inhibition and RhoA activation on basal and TGF β 1-stimulated SZP media accumulation. Primary, superficial zone articular chondrocytes were treated with (A) 10 and 50 μM Y27632, a competitive inhibitor

    Article Snippet: After the media exchange, chondrocytes were pretreated with an inhibitor for 1 h, before the addition of 3 ng/mL TGF β 1 (R & D Systems, Minneapolis, MN).

    Techniques: Inhibition, Activation Assay

    Immunoblot analysis of the expression of TGF-β1 and VEGF after 7, 14 and 21 days of the experiment in second-degree burns in rats in different treatment groups. C: untreated control; L: treated with 670-nm InGaP laser; P: treated with the Porophyllum ruderale extract PL: treated with the P. ruderale extract and 670-nm InGaP laser. Typical blots are shown above average densitometry results. Values are the mean and standard deviation of each group and were compared by ANOVA with Tukey’s post-test (* p

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Activity of Porophyllum ruderale leaf extract and 670-nm InGaP laser during burns repair in rats

    doi: 10.1186/s12906-015-0805-2

    Figure Lengend Snippet: Immunoblot analysis of the expression of TGF-β1 and VEGF after 7, 14 and 21 days of the experiment in second-degree burns in rats in different treatment groups. C: untreated control; L: treated with 670-nm InGaP laser; P: treated with the Porophyllum ruderale extract PL: treated with the P. ruderale extract and 670-nm InGaP laser. Typical blots are shown above average densitometry results. Values are the mean and standard deviation of each group and were compared by ANOVA with Tukey’s post-test (* p

    Article Snippet: Samples containing 50 μg protein were boiled for 5 min and submitted to 10 % (VEGF, 40 kDa) and 12 % (TGF-β1, 25 kDa) SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in a mini-gel apparatus (Mini-Protean®, Bio-Rad-Richmond, CA, USA).

    Techniques: Expressing, Standard Deviation

    NMuMG cells respond to both TGF-β3 WW and TGF-β3 WD . NMuMG cells were untreated (Un) or treated with TGF-β1 (T), BMP4 (B) or the indicated concentrations of TGF-β3 WW and TGF-β3 WD for 1 hr. Whole cell lysates were immunoblotted with the antibodies shown. Both TGF-β3 WW and TGF-β3 WD induce phosphorylation of pSMAD1/5, although the latter is less potent.

    Journal: eLife

    Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition

    doi: 10.7554/eLife.31756

    Figure Lengend Snippet: NMuMG cells respond to both TGF-β3 WW and TGF-β3 WD . NMuMG cells were untreated (Un) or treated with TGF-β1 (T), BMP4 (B) or the indicated concentrations of TGF-β3 WW and TGF-β3 WD for 1 hr. Whole cell lysates were immunoblotted with the antibodies shown. Both TGF-β3 WW and TGF-β3 WD induce phosphorylation of pSMAD1/5, although the latter is less potent.

    Article Snippet: Cells were treated with recombinant TGF-β1 (PeproTech, 100–21C; 2 ng/ml), BMP4 (PeproTech, 120-05ET; 20 ng/ml) and Noggin (PeproTech, 250–38; 300 ng/ml).

    Techniques:

    ID1 and ID3 are TGF-β-induced target genes that require the pSMAD1/5 signaling arm. ( A ) Western blots showing knockdown efficiency in MDA-MB-231s of the siRNAs shown. S3, SMAD3; S4, SMAD4; S1/5, SMAD1/5; NT, non targeting. Cells were untreated (U) or treated with TGF-β (T) or BMP4 (B) for 1 hr. Lysates were immunoblotted using the antibodies shown. ( B ) MDA-MB-231 cells were transfected with siRNAs against the indicated SMADs or a non-targeting control (NT) and then treated with TGF-β (T) or BMP4 (B) for 1 hr. Un, untreated. ( C ) MDA-MB-231 cells were left untreated or treated with TGF-β ± SB-431542 (SB; 0.25 µM or 10 µM) ± 1 µM LDN-193189 (LDN) or BMP4 ± 1 µM LDN-193189 for 1 hr. In B and C, gene expression was measured by qPCR. Data are presented as fold change relative to the untreated NT sample in ( B ) and to the (-) sample in ( C ) and are the means ± SEM of three independent experiments. Statistical significance is shown for selected comparisons. ( D ) NMuMG cells were treated with TGF-β or BMP4 ± the inhibitors indicated. Gene expression was measured by qPCR. The combination of 0.125 µM SB-431542 (SB) and 1 µM LDN-193189 (LDN) inhibited TGF-β-induced Id1 and Id3 expression without affecting JunB expression. The data are means ± SEM of at least two independent experiments. Statistical significance is shown for selected comparisons. ( E ) MDA-MB-231 cells were treated with TGF-β1 (T), BMP4 (B) or different concentrations of TGF-β3 WW and TGF-β3 WD for 1 hr as in Figure 3B . Gene expression was measured by qPCR. Both TGF-β3 WW and TGF-β3 WD led to the induction of ID1 , ID3 and JUNB , although the induction by TGF-β3 WD was weaker. A representative experiment of two, performed in triplicate is shown with means ± SD. 10.7554/eLife.31756.029 qPCR data for graphs in panel B. 10.7554/eLife.31756.030 qPCR data for graphs in panel C. 10.7554/eLife.31756.031 qPCR data for graphs in panel D. 10.7554/eLife.31756.032 qPCR data for graphs in panel E.

    Journal: eLife

    Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition

    doi: 10.7554/eLife.31756

    Figure Lengend Snippet: ID1 and ID3 are TGF-β-induced target genes that require the pSMAD1/5 signaling arm. ( A ) Western blots showing knockdown efficiency in MDA-MB-231s of the siRNAs shown. S3, SMAD3; S4, SMAD4; S1/5, SMAD1/5; NT, non targeting. Cells were untreated (U) or treated with TGF-β (T) or BMP4 (B) for 1 hr. Lysates were immunoblotted using the antibodies shown. ( B ) MDA-MB-231 cells were transfected with siRNAs against the indicated SMADs or a non-targeting control (NT) and then treated with TGF-β (T) or BMP4 (B) for 1 hr. Un, untreated. ( C ) MDA-MB-231 cells were left untreated or treated with TGF-β ± SB-431542 (SB; 0.25 µM or 10 µM) ± 1 µM LDN-193189 (LDN) or BMP4 ± 1 µM LDN-193189 for 1 hr. In B and C, gene expression was measured by qPCR. Data are presented as fold change relative to the untreated NT sample in ( B ) and to the (-) sample in ( C ) and are the means ± SEM of three independent experiments. Statistical significance is shown for selected comparisons. ( D ) NMuMG cells were treated with TGF-β or BMP4 ± the inhibitors indicated. Gene expression was measured by qPCR. The combination of 0.125 µM SB-431542 (SB) and 1 µM LDN-193189 (LDN) inhibited TGF-β-induced Id1 and Id3 expression without affecting JunB expression. The data are means ± SEM of at least two independent experiments. Statistical significance is shown for selected comparisons. ( E ) MDA-MB-231 cells were treated with TGF-β1 (T), BMP4 (B) or different concentrations of TGF-β3 WW and TGF-β3 WD for 1 hr as in Figure 3B . Gene expression was measured by qPCR. Both TGF-β3 WW and TGF-β3 WD led to the induction of ID1 , ID3 and JUNB , although the induction by TGF-β3 WD was weaker. A representative experiment of two, performed in triplicate is shown with means ± SD. 10.7554/eLife.31756.029 qPCR data for graphs in panel B. 10.7554/eLife.31756.030 qPCR data for graphs in panel C. 10.7554/eLife.31756.031 qPCR data for graphs in panel D. 10.7554/eLife.31756.032 qPCR data for graphs in panel E.

    Article Snippet: Cells were treated with recombinant TGF-β1 (PeproTech, 100–21C; 2 ng/ml), BMP4 (PeproTech, 120-05ET; 20 ng/ml) and Noggin (PeproTech, 250–38; 300 ng/ml).

    Techniques: Western Blot, Multiple Displacement Amplification, Transfection, Expressing, Real-time Polymerase Chain Reaction