recombinant human tgf β1  (R&D Systems)

 
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    Name:
    Recombinant Human TGF beta 1 Human Cell expressed CF
    Description:
    The Recombinant Human TGF beta 1 Human Cell expressed from R D Systems is derived from HEK293 The Recombinant Human TGF beta 1 Human Cell expressed has been validated for the following applications Bioactivity
    Catalog Number:
    7754-bh-025/cf
    Price:
    809
    Applications:
    Bioactivity
    Purity:
    >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
    Conjugate:
    Unconjugated
    Size:
    25 ug
    Category:
    Proteins and Enzymes
    Source:
    HEK293-derived Recombinant Human TGF-beta 1 (Human Cell-expressed)
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    Structured Review

    R&D Systems recombinant human tgf β1
    Schematic representation of proposed model for <t>TGF-β1-induced</t> activation of TAK1 in MMC. Under unstimulated conditions TAK1 interacts with TβRI through the complex formation with TAB2 and TRAF6. TGF-β1 stimulation-induced formation
    The Recombinant Human TGF beta 1 Human Cell expressed from R D Systems is derived from HEK293 The Recombinant Human TGF beta 1 Human Cell expressed has been validated for the following applications Bioactivity
    https://www.bioz.com/result/recombinant human tgf β1/product/R&D Systems
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    recombinant human tgf β1 - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Transforming Growth Factor-? (TGF-?1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-? Receptor Kinase Activity in Mesangial Cells *"

    Article Title: Transforming Growth Factor-? (TGF-?1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-? Receptor Kinase Activity in Mesangial Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.007146

    Schematic representation of proposed model for TGF-β1-induced activation of TAK1 in MMC. Under unstimulated conditions TAK1 interacts with TβRI through the complex formation with TAB2 and TRAF6. TGF-β1 stimulation-induced formation
    Figure Legend Snippet: Schematic representation of proposed model for TGF-β1-induced activation of TAK1 in MMC. Under unstimulated conditions TAK1 interacts with TβRI through the complex formation with TAB2 and TRAF6. TGF-β1 stimulation-induced formation

    Techniques Used: Activation Assay

    TβRI-mediated and TGF-β1-induced TAK1 phosphorylation requires its own kinase activity and TAB1. A , TAB1 induces autophosphorylation of TAK1. FLAG-TAB1 was coexpressed in MMC with WT, kinase-deficient mutant ( KD ; K63W), or phosphorylation
    Figure Legend Snippet: TβRI-mediated and TGF-β1-induced TAK1 phosphorylation requires its own kinase activity and TAB1. A , TAB1 induces autophosphorylation of TAK1. FLAG-TAB1 was coexpressed in MMC with WT, kinase-deficient mutant ( KD ; K63W), or phosphorylation

    Techniques Used: Activity Assay, Mutagenesis

    TRAF6 mediates the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation. A , knockdown of TRAF6 by siRNA inhibits TβRI-TAK1 interaction. After transfection of MMC with control siRNA (−) or siRNA specific for
    Figure Legend Snippet: TRAF6 mediates the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation. A , knockdown of TRAF6 by siRNA inhibits TβRI-TAK1 interaction. After transfection of MMC with control siRNA (−) or siRNA specific for

    Techniques Used: Activation Assay, Transfection

    2) Product Images from "Downregulation of delta‐aminolevulinate dehydratase is associated with poor prognosis in patients with breast cancer"

    Article Title: Downregulation of delta‐aminolevulinate dehydratase is associated with poor prognosis in patients with breast cancer

    Journal: Cancer Science

    doi: 10.1111/cas.13180

    Delta‐aminolevulinate dehydratase ( ALAD ) regulates transforming growth factor‐β ( TGF ‐β)‐mediated epithelial–mesenchymal transition in MCF 10A cells. (a,b) RT –quantitative PCR analysis of ALAD expression levels in MCF 10A cells treated with TGF ‐β1 at indicated concentrations (a) and time (b) compared to the control. (c) Morphology of MCF 10A cells with Control and ALAD after 7 days of treatment with TGF ‐β1 (10 ng/ mL ) in MCF 10A cells. (d) TGF ‐β signaling activity determined by luciferase report assay. (e,f) Western blot of E‐cadherin, vimentin, and ALAD expression in indicated MCF 10A cells. (g) Invasive ability of indicated MCF 10A cells. * P
    Figure Legend Snippet: Delta‐aminolevulinate dehydratase ( ALAD ) regulates transforming growth factor‐β ( TGF ‐β)‐mediated epithelial–mesenchymal transition in MCF 10A cells. (a,b) RT –quantitative PCR analysis of ALAD expression levels in MCF 10A cells treated with TGF ‐β1 at indicated concentrations (a) and time (b) compared to the control. (c) Morphology of MCF 10A cells with Control and ALAD after 7 days of treatment with TGF ‐β1 (10 ng/ mL ) in MCF 10A cells. (d) TGF ‐β signaling activity determined by luciferase report assay. (e,f) Western blot of E‐cadherin, vimentin, and ALAD expression in indicated MCF 10A cells. (g) Invasive ability of indicated MCF 10A cells. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Luciferase, Western Blot

    3) Product Images from "Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene"

    Article Title: Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0020054

    Semi-Quantitative RT-PCR Analysis of SmGCP mRNA The bottom panel shows the agarose gel separation of the PCR products of SmGCP (bottom), and the constitutively transcribed control, α-tubulin (top). Panel A: Lanes are numbered and the respective stages are listed at the bottom of the panel. Panel B: Adult worm pairs (42-d-old) were left untreated (lane 1) or treated with human TGF-β1 (1 nM; lane 2) or human BMP2 (5 nM; lane 3). Top of each panel shows a bar graph representation of the relative PCR band intensities (%) of SmGCP compared to that of α-tubulin control. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).
    Figure Legend Snippet: Semi-Quantitative RT-PCR Analysis of SmGCP mRNA The bottom panel shows the agarose gel separation of the PCR products of SmGCP (bottom), and the constitutively transcribed control, α-tubulin (top). Panel A: Lanes are numbered and the respective stages are listed at the bottom of the panel. Panel B: Adult worm pairs (42-d-old) were left untreated (lane 1) or treated with human TGF-β1 (1 nM; lane 2) or human BMP2 (5 nM; lane 3). Top of each panel shows a bar graph representation of the relative PCR band intensities (%) of SmGCP compared to that of α-tubulin control. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).

    Techniques Used: Quantitative RT-PCR, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Standard Deviation

    Silencing of TGF-β–Induced Expression of SmGCP by Knocking Down SmTβRII Expression Semi-quantitative RT-PCR analyses for transcripts of SmGCP as well as various components of schistosomal TGF-β signaling pathways in 35-d-old and 28-d-old old worm pairs, untransformed and transformed with SmTβRII-siRNA, and either left untreated or treated with TGF-β1 (1 nM). The top panel shows the agarose gel separation of the PCR products of SmTβRII (panel B), SmTβRI (panel C), SmGCP (panel D), SmSmad4 (panel E), SmSmad2 (panel F), SmSmad1 (panel G), and the constitutively transcribed control, α-tubulin (panel A). The lanes are labeled to show detailed treatment of each sample. The bar graph representation shows the percentage values of the optical densities in pixels of the PCR bands for each gene compared to the corresponding band of α-tubulin control from the same stage. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).
    Figure Legend Snippet: Silencing of TGF-β–Induced Expression of SmGCP by Knocking Down SmTβRII Expression Semi-quantitative RT-PCR analyses for transcripts of SmGCP as well as various components of schistosomal TGF-β signaling pathways in 35-d-old and 28-d-old old worm pairs, untransformed and transformed with SmTβRII-siRNA, and either left untreated or treated with TGF-β1 (1 nM). The top panel shows the agarose gel separation of the PCR products of SmTβRII (panel B), SmTβRI (panel C), SmGCP (panel D), SmSmad4 (panel E), SmSmad2 (panel F), SmSmad1 (panel G), and the constitutively transcribed control, α-tubulin (panel A). The lanes are labeled to show detailed treatment of each sample. The bar graph representation shows the percentage values of the optical densities in pixels of the PCR bands for each gene compared to the corresponding band of α-tubulin control from the same stage. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).

    Techniques Used: Expressing, Quantitative RT-PCR, Transformation Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Labeling, Standard Deviation

    The Transduction of TGF-β Signal to SmSmad2 via Activated SmTβRII/SmTβRI Receptor Complex, In Vitro: Co-immunoprecipitation of SmSmad2-MH2 and SmSmad4 35 S-labeled, in vitro translated products of SmSmad2-MH2 (panel A) and SmSmad2-MH2/AAA (panel B) were incubated with SmSmad4 in the presence of SmTβR-I (wt) and SmTβR-II in the presence or absence of TGF-β1 (1.0 nM) or BMP2 (5.0 nM). Radiolabeled, in vitro translated products were co-precipitated with SmSmad4, using anti-SmSmad4-linker IgG and Protein A Sepharose beads (Amersham Biosciences). Background precipitation was removed by treating 35 S-labeled in vitro translated products with anti-SmSmad4-linker IgG and Protein A Sepharose beads. The pre-cleared lysates were then used in the above-described reactions. A positive control reaction (lane 3) was included, in which SmSmad2-MH2, or the AAA mutant peptide, were reacted with SmSmad4 in the presence of the active mutant form of type I receptor, SmTβR-I (Q-D). Reactions, which contain either SmSmad2-MH2 or its AAA mutant form with SmSmad4 in the presence of wild-type SmTβRI, represent the negative controls of the assay (lane 4). Immunoprecipitated products were separated by SDS-PAGE and subjected to autofluorography. Lanes are labeled to specify the input components of each reaction. In vitro translated products (20% of input) are shown (lane 1). Percentage values of precipitated reactive radiolabeled product of each reaction are shown at the bottom of each lane.
    Figure Legend Snippet: The Transduction of TGF-β Signal to SmSmad2 via Activated SmTβRII/SmTβRI Receptor Complex, In Vitro: Co-immunoprecipitation of SmSmad2-MH2 and SmSmad4 35 S-labeled, in vitro translated products of SmSmad2-MH2 (panel A) and SmSmad2-MH2/AAA (panel B) were incubated with SmSmad4 in the presence of SmTβR-I (wt) and SmTβR-II in the presence or absence of TGF-β1 (1.0 nM) or BMP2 (5.0 nM). Radiolabeled, in vitro translated products were co-precipitated with SmSmad4, using anti-SmSmad4-linker IgG and Protein A Sepharose beads (Amersham Biosciences). Background precipitation was removed by treating 35 S-labeled in vitro translated products with anti-SmSmad4-linker IgG and Protein A Sepharose beads. The pre-cleared lysates were then used in the above-described reactions. A positive control reaction (lane 3) was included, in which SmSmad2-MH2, or the AAA mutant peptide, were reacted with SmSmad4 in the presence of the active mutant form of type I receptor, SmTβR-I (Q-D). Reactions, which contain either SmSmad2-MH2 or its AAA mutant form with SmSmad4 in the presence of wild-type SmTβRI, represent the negative controls of the assay (lane 4). Immunoprecipitated products were separated by SDS-PAGE and subjected to autofluorography. Lanes are labeled to specify the input components of each reaction. In vitro translated products (20% of input) are shown (lane 1). Percentage values of precipitated reactive radiolabeled product of each reaction are shown at the bottom of each lane.

    Techniques Used: Transduction, In Vitro, Immunoprecipitation, Labeling, Incubation, Positive Control, Mutagenesis, SDS Page

    4) Product Images from "TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145523

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: Isolation, Translocation Assay

    5) Product Images from "EGF antagonizes TGF-?-induced tropoelastin expression in lung fibroblasts via stabilization of Smad corepressor TGIF"

    Article Title: EGF antagonizes TGF-?-induced tropoelastin expression in lung fibroblasts via stabilization of Smad corepressor TGIF

    Journal:

    doi: 10.1152/ajplung.00289.2007

    EGF does not decrease TGF-β-induced nuclear accumulation of Smad2/3 in RFL-6 fibroblasts. A : postconfluent serum-starved cells were treated for 1 h with or without 10 ng/ml EGF, 2 ng/ml TGF-β1, or EGF + TGF-β1. Cytosolic
    Figure Legend Snippet: EGF does not decrease TGF-β-induced nuclear accumulation of Smad2/3 in RFL-6 fibroblasts. A : postconfluent serum-starved cells were treated for 1 h with or without 10 ng/ml EGF, 2 ng/ml TGF-β1, or EGF + TGF-β1. Cytosolic

    Techniques Used:

    6) Product Images from "Upregulation of Retinal Dehydrogenase 2 in Alternatively Activated Macrophages during Retinoid-dependent Type-2 Immunity to Helminth Infection in Mice"

    Article Title: Upregulation of Retinal Dehydrogenase 2 in Alternatively Activated Macrophages during Retinoid-dependent Type-2 Immunity to Helminth Infection in Mice

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002883

    AAMφ have an enhanced capacity to induce Foxp3 expression in T cells. ( A ) Flow cytometric analysis of aldehyde dehydrogenase (ALDH) activity in peritoneal cells using the Aldefluor assay. Representative contour plots are gated on live F4/80 + CD11b + cells. ( B ) Flow cytometric analysis of activated (anti-CD3+IL-2) labeled naïve CD4 + cells (gated on CD11b − , CD3 + , live Hoechst negative, CD4 + cells) cultured with peritoneal macrophages from control untreated mice (Res. PEC), mice injected with Thioglycollate (Thio.) or mice injected with Thioglycollate and IL-4 (Thio.+IL-4). Gates show the frequency of cells that have undergone more than 2 rounds of cell division (at Day 3) or more than 4 rounds of cell division (at Day 6). ( C ) Flow cytometry histogram plots showing the percentage of co-cultured CD4 + cells expressing Foxp3 (at Day 6) when activated with anti-CD3 and IL-2 alone (Control) or with exogenous TGF-β1 (2 ng/ml) or TGF-β1 and RA (100 nm). ( D ) Flow cytometry contour plots showing the percentage of CD25 + , Foxp3 + CD4 + T cells after 6 days of co-culture with the RA inhibitor LE540 (1 µM). ( E ) Flow cytometry contour plots showing the effects of TGF-β1 and LE540 on the expression of α4β7 integrin and Foxp3 in co-cultured (Day 6) CD4 + cells. (F) Flow cytometry histogram plots showing the induction of CCR9 by TGF-β1 and RA (solid grey) when compared to CD4 + cells cultured with anti-CD3 and IL-2 alone (Control, dashed line) or with the addition of TGF-β1 only (solid thick line). Results are all representative of two or three independent experiments.
    Figure Legend Snippet: AAMφ have an enhanced capacity to induce Foxp3 expression in T cells. ( A ) Flow cytometric analysis of aldehyde dehydrogenase (ALDH) activity in peritoneal cells using the Aldefluor assay. Representative contour plots are gated on live F4/80 + CD11b + cells. ( B ) Flow cytometric analysis of activated (anti-CD3+IL-2) labeled naïve CD4 + cells (gated on CD11b − , CD3 + , live Hoechst negative, CD4 + cells) cultured with peritoneal macrophages from control untreated mice (Res. PEC), mice injected with Thioglycollate (Thio.) or mice injected with Thioglycollate and IL-4 (Thio.+IL-4). Gates show the frequency of cells that have undergone more than 2 rounds of cell division (at Day 3) or more than 4 rounds of cell division (at Day 6). ( C ) Flow cytometry histogram plots showing the percentage of co-cultured CD4 + cells expressing Foxp3 (at Day 6) when activated with anti-CD3 and IL-2 alone (Control) or with exogenous TGF-β1 (2 ng/ml) or TGF-β1 and RA (100 nm). ( D ) Flow cytometry contour plots showing the percentage of CD25 + , Foxp3 + CD4 + T cells after 6 days of co-culture with the RA inhibitor LE540 (1 µM). ( E ) Flow cytometry contour plots showing the effects of TGF-β1 and LE540 on the expression of α4β7 integrin and Foxp3 in co-cultured (Day 6) CD4 + cells. (F) Flow cytometry histogram plots showing the induction of CCR9 by TGF-β1 and RA (solid grey) when compared to CD4 + cells cultured with anti-CD3 and IL-2 alone (Control, dashed line) or with the addition of TGF-β1 only (solid thick line). Results are all representative of two or three independent experiments.

    Techniques Used: Expressing, Flow Cytometry, Activity Assay, Labeling, Cell Culture, Mouse Assay, Injection, Cytometry, Co-Culture Assay

    7) Product Images from "Transcriptional Regulation of Seprase in Invasive Melanoma Cells by Transforming Growth Factor-β Signaling *"

    Article Title: Transcriptional Regulation of Seprase in Invasive Melanoma Cells by Transforming Growth Factor-β Signaling *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.568501

    TGF-β1 increases endogenous seprase mRNA levels and seprase promoter activity in invasive melanoma cell lines. A , the cell lines indicated were serum-starved for 8 h and then untreated or treated with 2 ng/ml TGF-β1 over a 24-h time course. Relative seprase mRNA levels were determined by qRT-PCR for all time points as indicated ( top panel ). Seprase mRNA levels were normalized to β-actin levels for all samples for comparison. Results are the mean ± S.D. of triplicate samples from two of at least three experiments ( n = 6; *, p
    Figure Legend Snippet: TGF-β1 increases endogenous seprase mRNA levels and seprase promoter activity in invasive melanoma cell lines. A , the cell lines indicated were serum-starved for 8 h and then untreated or treated with 2 ng/ml TGF-β1 over a 24-h time course. Relative seprase mRNA levels were determined by qRT-PCR for all time points as indicated ( top panel ). Seprase mRNA levels were normalized to β-actin levels for all samples for comparison. Results are the mean ± S.D. of triplicate samples from two of at least three experiments ( n = 6; *, p

    Techniques Used: Activity Assay, Quantitative RT-PCR

    Assessment of Smad3/4 and c-Ski binding to the TGF-β-responsive element of the human seprase promoter in invasive and non-invasive cells. A , A375 melanoma cells were serum-starved for 8 h followed by treatment with or without 5 ng/ml TGF-β1 in CCC media for an additional 8 h and then collected for ChIP analysis. Antibodies used for ChIP were anti-Smad2/3, anti-Smad4, ant-c-Ski, and anti-IgG. Promoter regions examined were putative SBE site 5 (low TGF-β responsiveness), putative SBE site 7 (high TGF-β responsiveness), and non-site (negative control; distal promoter region). Enrichment was assessed by real time PCR and is represented as percentage of input. Results are the mean ± S.D. of triplicate samples from two of three independent experiments. Error bars represent S.D. B , same as A but using the LOX cell line. C , same as A but using the SK-MEL-28 cell line. D , same as A but using primary HEMa-LP cells.
    Figure Legend Snippet: Assessment of Smad3/4 and c-Ski binding to the TGF-β-responsive element of the human seprase promoter in invasive and non-invasive cells. A , A375 melanoma cells were serum-starved for 8 h followed by treatment with or without 5 ng/ml TGF-β1 in CCC media for an additional 8 h and then collected for ChIP analysis. Antibodies used for ChIP were anti-Smad2/3, anti-Smad4, ant-c-Ski, and anti-IgG. Promoter regions examined were putative SBE site 5 (low TGF-β responsiveness), putative SBE site 7 (high TGF-β responsiveness), and non-site (negative control; distal promoter region). Enrichment was assessed by real time PCR and is represented as percentage of input. Results are the mean ± S.D. of triplicate samples from two of three independent experiments. Error bars represent S.D. B , same as A but using the LOX cell line. C , same as A but using the SK-MEL-28 cell line. D , same as A but using primary HEMa-LP cells.

    Techniques Used: Binding Assay, Countercurrent Chromatography, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

    Functional analysis of putative SBEs within the human seprase promoter. A , site-directed mutagenesis was carried out to mutate all nine putative SBEs in the 674-bp (−469/+205) seprase promoter. To assess the transcriptional activity of resultant mutants, LOX cells were transfected with either the pGL4-Sep674 (WT) reporter construct or pGL4-Sep674 harboring individual mutations in SBEs 1–9, and luciferase assays were conducted. Controls were pGL4-Sep674 mNon-site and pGL4 vector ( Vect ). All samples were normalized for transfection efficiency by co-transfection with pRL-TK vector. Reporter activity is expressed as relative luciferase units ( RLUs ). Results are the mean ± S.D. of triplicate samples from one (representative) of three independent experiments. Error bars represent S.D. B , same as for A with the exception that only SBE mutants 5, 6, and 7 or double/triple/quadruple mutants of these SBEs were compared with WT. C , to map out the TGF-β-responsive region of the seprase promoter, LOX cells were transfected with either the pGL4-Sep674 (WT) reporter plasmid or SBE mutant 5, 6, or 7 or multiple SBE mutant versions. Controls were pGL4-Sep674 mNon-site and pGL4 vector. Cells were untreated or treated with 2 ng/ml TGF-β1 for 12 h before luciferase activities were determined. Samples were normalized for transfection efficiency by co-transfection with pRL-TK vector. Reporter activity is expressed as relative luciferase units ( RLUs ). Results are the mean ± S.D. of triplicate samples from one (representative) of three independent experiments. Error bars represent S.D.
    Figure Legend Snippet: Functional analysis of putative SBEs within the human seprase promoter. A , site-directed mutagenesis was carried out to mutate all nine putative SBEs in the 674-bp (−469/+205) seprase promoter. To assess the transcriptional activity of resultant mutants, LOX cells were transfected with either the pGL4-Sep674 (WT) reporter construct or pGL4-Sep674 harboring individual mutations in SBEs 1–9, and luciferase assays were conducted. Controls were pGL4-Sep674 mNon-site and pGL4 vector ( Vect ). All samples were normalized for transfection efficiency by co-transfection with pRL-TK vector. Reporter activity is expressed as relative luciferase units ( RLUs ). Results are the mean ± S.D. of triplicate samples from one (representative) of three independent experiments. Error bars represent S.D. B , same as for A with the exception that only SBE mutants 5, 6, and 7 or double/triple/quadruple mutants of these SBEs were compared with WT. C , to map out the TGF-β-responsive region of the seprase promoter, LOX cells were transfected with either the pGL4-Sep674 (WT) reporter plasmid or SBE mutant 5, 6, or 7 or multiple SBE mutant versions. Controls were pGL4-Sep674 mNon-site and pGL4 vector. Cells were untreated or treated with 2 ng/ml TGF-β1 for 12 h before luciferase activities were determined. Samples were normalized for transfection efficiency by co-transfection with pRL-TK vector. Reporter activity is expressed as relative luciferase units ( RLUs ). Results are the mean ± S.D. of triplicate samples from one (representative) of three independent experiments. Error bars represent S.D.

    Techniques Used: Functional Assay, Mutagenesis, Activity Assay, Transfection, Construct, Luciferase, Plasmid Preparation, Cotransfection

    8) Product Images from "Pin1 Protein Regulates Smad Protein Signaling and Pulmonary Fibrosis *"

    Article Title: Pin1 Protein Regulates Smad Protein Signaling and Pulmonary Fibrosis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.313684

    Pin knockout suppresses TGF-β1-Smad signaling in primary lung Fb. A , cells were infected with lentiviral vectors containing Smad2/3-responsive luciferase reporter and Renilla -luciferase constitutive reporter. After 2 days, cultures were treated
    Figure Legend Snippet: Pin knockout suppresses TGF-β1-Smad signaling in primary lung Fb. A , cells were infected with lentiviral vectors containing Smad2/3-responsive luciferase reporter and Renilla -luciferase constitutive reporter. After 2 days, cultures were treated

    Techniques Used: Knock-Out, Infection, Luciferase

    Smad6 knockdown restores TGF-β signaling in Pin1 null cells. A , primary lung Fb transfected with control or Smad6-specific siRNA (50 n m ) were treated with TGF-β1 for 1 h. Whole cell lysates were immunoblotted with antibodies shown on the
    Figure Legend Snippet: Smad6 knockdown restores TGF-β signaling in Pin1 null cells. A , primary lung Fb transfected with control or Smad6-specific siRNA (50 n m ) were treated with TGF-β1 for 1 h. Whole cell lysates were immunoblotted with antibodies shown on the

    Techniques Used: Transfection

    Pin1 knockout decreases type III collagen expression in primary lung Fb. A , wild-type and Pin1 −/− primary Fb were starved for 2 days before stimulation with TGF-β1 (1 ng/ml) for 12 h. Total RNA were subjected to qPCR analysis.
    Figure Legend Snippet: Pin1 knockout decreases type III collagen expression in primary lung Fb. A , wild-type and Pin1 −/− primary Fb were starved for 2 days before stimulation with TGF-β1 (1 ng/ml) for 12 h. Total RNA were subjected to qPCR analysis.

    Techniques Used: Knock-Out, Expressing, Real-time Polymerase Chain Reaction

    9) Product Images from "TIMP-1 mediates TGF-β-dependent crosstalk between hepatic stellate and cancer cells via FAK signaling"

    Article Title: TIMP-1 mediates TGF-β-dependent crosstalk between hepatic stellate and cancer cells via FAK signaling

    Journal: Scientific Reports

    doi: 10.1038/srep16492

    Suppression of TGF-β-Induced EMT and Akt Signaling In Vitro by TGF-β Blockade. ( A ) Effects of TGF-β1 on the proliferation of SK-HEP1, SNU354, and HepG2 cells. Cells were treated with the indicated concentration of TGF-β1 for 72 h. ( B ) Effects of EW-7197 on the proliferation of SK-HEP1, SNU354, and HepG2 cells. Cells were treated with the indicated concentration of EW-7197 for 72 h. ( C ) Effects of EW-7197 on the protein expression levels of fibronectin (FN), N-cadherin (N-CAD), and CTGF in SK-HEP1, SNU354, and HepG2 cells. Cells were treated with the indicated concentration of EW-7197 in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. GAPDH was used as a reference. ( D ) Effects of EW-7197 (1 μM) on the migration of SK-HEP1 cells. Cells were treated with EW-7197 in the presence or absence of TGF-β1 (2 ng/ml) for 30 h. Scale bars: 200 μm. ( E ) Effects of EW-7197 on AKT phosphorylation in SK-HEP1, SNU354, and HepG2 cells. Cells were treated with the indicated concentration of EW-7197 in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. AKT was used as a reference. ( F ) Effects of EW-7197 (1 μM) and Akt inhibitor (10 μM, AKTI) on the anchorage-independent growth of SK-HEP1 cells. Cells were treated with the indicated drug in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. After treatment, the cells were counted and placed in soft agar for colony assay to determine cell survival. *** p
    Figure Legend Snippet: Suppression of TGF-β-Induced EMT and Akt Signaling In Vitro by TGF-β Blockade. ( A ) Effects of TGF-β1 on the proliferation of SK-HEP1, SNU354, and HepG2 cells. Cells were treated with the indicated concentration of TGF-β1 for 72 h. ( B ) Effects of EW-7197 on the proliferation of SK-HEP1, SNU354, and HepG2 cells. Cells were treated with the indicated concentration of EW-7197 for 72 h. ( C ) Effects of EW-7197 on the protein expression levels of fibronectin (FN), N-cadherin (N-CAD), and CTGF in SK-HEP1, SNU354, and HepG2 cells. Cells were treated with the indicated concentration of EW-7197 in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. GAPDH was used as a reference. ( D ) Effects of EW-7197 (1 μM) on the migration of SK-HEP1 cells. Cells were treated with EW-7197 in the presence or absence of TGF-β1 (2 ng/ml) for 30 h. Scale bars: 200 μm. ( E ) Effects of EW-7197 on AKT phosphorylation in SK-HEP1, SNU354, and HepG2 cells. Cells were treated with the indicated concentration of EW-7197 in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. AKT was used as a reference. ( F ) Effects of EW-7197 (1 μM) and Akt inhibitor (10 μM, AKTI) on the anchorage-independent growth of SK-HEP1 cells. Cells were treated with the indicated drug in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. After treatment, the cells were counted and placed in soft agar for colony assay to determine cell survival. *** p

    Techniques Used: In Vitro, Concentration Assay, Expressing, Migration, Colony Assay

    Secreted TIMP-1 Mediates Crosstalk between HSC and HCC Cells. ( A ) TIMP-1 mRNA levels in LX-2 cells. Cells were treated with EW-7197 (1 μM) in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. HPRT was used as a reference. ( B ) TIMP-1 protein levels in CM. The GADPH level in an aliquot of total cell lysate was used as a reference. ( C ) mRNA expression levels of TIMP-1 in HCC patients from the Chen and Mas dataset from Oncomine (www.oncomine.com). ( D ) Effects of TIMP-1 on the proliferation of SK-HEP1 cells. Cells were treated with CM for 72 h. ( E ) Representative images (left) and densitometric analysis (right) of wound-healing assay of SK-HEP1 cells. Cells were treated with CM for 30 h. Scale bars: 200 μm. ( F ) Representative images (left) and densitometric analysis (right) of soft agar assay of SK-HEP1 cells. Cells were treated with CM for 24 h. After treatment, the cells were counted and placed in soft agar for colony assay to determine cell survival. *** p
    Figure Legend Snippet: Secreted TIMP-1 Mediates Crosstalk between HSC and HCC Cells. ( A ) TIMP-1 mRNA levels in LX-2 cells. Cells were treated with EW-7197 (1 μM) in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. HPRT was used as a reference. ( B ) TIMP-1 protein levels in CM. The GADPH level in an aliquot of total cell lysate was used as a reference. ( C ) mRNA expression levels of TIMP-1 in HCC patients from the Chen and Mas dataset from Oncomine (www.oncomine.com). ( D ) Effects of TIMP-1 on the proliferation of SK-HEP1 cells. Cells were treated with CM for 72 h. ( E ) Representative images (left) and densitometric analysis (right) of wound-healing assay of SK-HEP1 cells. Cells were treated with CM for 30 h. Scale bars: 200 μm. ( F ) Representative images (left) and densitometric analysis (right) of soft agar assay of SK-HEP1 cells. Cells were treated with CM for 24 h. After treatment, the cells were counted and placed in soft agar for colony assay to determine cell survival. *** p

    Techniques Used: Expressing, Wound Healing Assay, Soft Agar Assay, Colony Assay

    10) Product Images from "?-arrestin Deficiency Protects Against Pulmonary Fibrosis in Mice and Prevents Fibroblast Invasion of Extracellular Matrix"

    Article Title: ?-arrestin Deficiency Protects Against Pulmonary Fibrosis in Mice and Prevents Fibroblast Invasion of Extracellular Matrix

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.3001564

    Primary lung fibroblasts from β-arrestin1 −/− and β-arrestin2 −/− mice exhibit a normal response to TGF-β stimulation. (A–B) Fibroblasts were treated with 10 ng/ml ( A ) or 5 ng/ml ( B ) of TGF-β1
    Figure Legend Snippet: Primary lung fibroblasts from β-arrestin1 −/− and β-arrestin2 −/− mice exhibit a normal response to TGF-β stimulation. (A–B) Fibroblasts were treated with 10 ng/ml ( A ) or 5 ng/ml ( B ) of TGF-β1

    Techniques Used: Mouse Assay

    11) Product Images from "Rb/E2F4 and Smad2/3 Link Survivin to TGF-?-induced Apoptosis and Tumor Progression"

    Article Title: Rb/E2F4 and Smad2/3 Link Survivin to TGF-?-induced Apoptosis and Tumor Progression

    Journal: Oncogene

    doi: 10.1038/onc.2008.165

    Cellular survivin levels control the sensitivity to TGF-β and chemotherapeutic drugs (A) NRP-154 cells plated in GM3 were infected overnight with 1/500 dilution of the stock control (CTL) or survivin (Suv) adenoviruses, and then treated with vehicle or TGF-β1 (1 ng/ml) for 48 h. Cells were analyzed for DNA content by propidium iodide staining and FACS. The percentage of cells with hypodiploid DNA content (Sub-G1) is indicated and plotted. (B, C) Capase-3 activation was monitored by Western blot analysis of cleaved form of caspase-3 (Casp-3, active). NRP-154 cells were infected in the same way as in (A) with control (Ad-CTL) or survivin (Ad-Suv) adenoviruses, and then treated with vehicle or 10 ng/ml TGF-β (B); Etoposide (50 µM), Taxol (5 nM) or 0.1% DMSO (C) for 40 h. The level of overexpressed survivin was also shown. (D) NRP-154 cells, after virus infection, were treated with Taxol (2 nM) and/or TGF-β1 (0.3 ng/ml) for 40 h and assayed for caspase-3 cleavage. (E) NRP-154 cells expressing doxycycline-inducible shRNA targeting LacZ (sh-LacZ) or survivin (sh-Suv) were plated in GM2.1 medium containing 0.1 µg/ml of doxycycline. Two days later cells were either harvested to assay for survivin protein silencing (top) or treated with vehicle, TGF-β1 (10 ng/ml), DMSO (0.1%), etoposide (25 µM), or taxol (2 nM) for 40 h to assay active caspase-3 by Western blot (bottom).
    Figure Legend Snippet: Cellular survivin levels control the sensitivity to TGF-β and chemotherapeutic drugs (A) NRP-154 cells plated in GM3 were infected overnight with 1/500 dilution of the stock control (CTL) or survivin (Suv) adenoviruses, and then treated with vehicle or TGF-β1 (1 ng/ml) for 48 h. Cells were analyzed for DNA content by propidium iodide staining and FACS. The percentage of cells with hypodiploid DNA content (Sub-G1) is indicated and plotted. (B, C) Capase-3 activation was monitored by Western blot analysis of cleaved form of caspase-3 (Casp-3, active). NRP-154 cells were infected in the same way as in (A) with control (Ad-CTL) or survivin (Ad-Suv) adenoviruses, and then treated with vehicle or 10 ng/ml TGF-β (B); Etoposide (50 µM), Taxol (5 nM) or 0.1% DMSO (C) for 40 h. The level of overexpressed survivin was also shown. (D) NRP-154 cells, after virus infection, were treated with Taxol (2 nM) and/or TGF-β1 (0.3 ng/ml) for 40 h and assayed for caspase-3 cleavage. (E) NRP-154 cells expressing doxycycline-inducible shRNA targeting LacZ (sh-LacZ) or survivin (sh-Suv) were plated in GM2.1 medium containing 0.1 µg/ml of doxycycline. Two days later cells were either harvested to assay for survivin protein silencing (top) or treated with vehicle, TGF-β1 (10 ng/ml), DMSO (0.1%), etoposide (25 µM), or taxol (2 nM) for 40 h to assay active caspase-3 by Western blot (bottom).

    Techniques Used: Infection, CTL Assay, Staining, FACS, Activation Assay, Western Blot, Expressing, shRNA

    TGF-β downregulates survivin levels through a transcriptional mechanism (A) NRP-154 cells plated in GM3 medium were treated the next day with vehicle or 10 ng/ml of TGF-β1 or BMP-4 for the indicated time. P-Smad levels that are controls for the activation of downstream signals by these ligands were measured by Western blot analyses. Note that P-Smad3 antibody recognizes both P-Smad3 and P-Smad1, 5, 8 as indicated by the arrows. (B and C) NRP-154 cells were treated with TGF-β1 (10ng/ml) for the indicated time or with various doses of TGF-β1 for 16 h. Samples were subjected to Western blot (B) or RT-PCR (C). (D) NRP-154 cells were plated in GM2.1 medium and transfected with 0.4 µg of Suv-829 or empty luciferase vector (EV), 20 ng of CMV-driven renilla vector and 0.6 µg of pcDNA3. TGF-β1 (10 ng/ml) or vehicle was added 24 h after transfection, and cells were incubated for an additional 24 h before assay. Firefly luciferase values were normalized to renilla luciferase.
    Figure Legend Snippet: TGF-β downregulates survivin levels through a transcriptional mechanism (A) NRP-154 cells plated in GM3 medium were treated the next day with vehicle or 10 ng/ml of TGF-β1 or BMP-4 for the indicated time. P-Smad levels that are controls for the activation of downstream signals by these ligands were measured by Western blot analyses. Note that P-Smad3 antibody recognizes both P-Smad3 and P-Smad1, 5, 8 as indicated by the arrows. (B and C) NRP-154 cells were treated with TGF-β1 (10ng/ml) for the indicated time or with various doses of TGF-β1 for 16 h. Samples were subjected to Western blot (B) or RT-PCR (C). (D) NRP-154 cells were plated in GM2.1 medium and transfected with 0.4 µg of Suv-829 or empty luciferase vector (EV), 20 ng of CMV-driven renilla vector and 0.6 µg of pcDNA3. TGF-β1 (10 ng/ml) or vehicle was added 24 h after transfection, and cells were incubated for an additional 24 h before assay. Firefly luciferase values were normalized to renilla luciferase.

    Techniques Used: Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation, Incubation

    TGF-β type I receptor kinase activity is essential for the repression of survivin by TGF-β (A) Top: NRP-154 cells were treated in GM3 medium with 0.1% DMSO or 10 µM of SB431542 for 2h, followed by 30 min treatment with or without 10 ng/ml of TGF-β1. Bottom: NRP-154 cells were seeded at a density of 10 4 cells/ml/12-well plate in GM3 medium and treated with 0.1% DMSO or 10 µM of SB431542 24 h prior to TGF-β1 (10 ng/ml, 48 h). To determine cell number, cells detached with trypsinization were enumerated using a Coulter Counter (Coulter Electronics). (B) NRP-154 cells plated in GM3 medium were treated with 0.1% DMSO or 10 µM of SB431542 for 1h, followed by treatment with vehicle or 10 ng/ml of TGF-β1 for 24 h, and expression of survivin was analyzed by Western blot. (C) NRP-154 cells transfected with 0.4 µg of Suv-829 luciferase reporter construct were treated the same way as in (B), and then cells were harvested for luciferase assay. (D) NRP-154 cells were infected in GM3 with different doses (indicated as the dilution fold above the brackets) of stock adenoviruses expressing control (C) or HA-CA-ALK5 (A) for 5 h. Whole cell lysates were collected after an additional 29 h and analyzed by Western blot.
    Figure Legend Snippet: TGF-β type I receptor kinase activity is essential for the repression of survivin by TGF-β (A) Top: NRP-154 cells were treated in GM3 medium with 0.1% DMSO or 10 µM of SB431542 for 2h, followed by 30 min treatment with or without 10 ng/ml of TGF-β1. Bottom: NRP-154 cells were seeded at a density of 10 4 cells/ml/12-well plate in GM3 medium and treated with 0.1% DMSO or 10 µM of SB431542 24 h prior to TGF-β1 (10 ng/ml, 48 h). To determine cell number, cells detached with trypsinization were enumerated using a Coulter Counter (Coulter Electronics). (B) NRP-154 cells plated in GM3 medium were treated with 0.1% DMSO or 10 µM of SB431542 for 1h, followed by treatment with vehicle or 10 ng/ml of TGF-β1 for 24 h, and expression of survivin was analyzed by Western blot. (C) NRP-154 cells transfected with 0.4 µg of Suv-829 luciferase reporter construct were treated the same way as in (B), and then cells were harvested for luciferase assay. (D) NRP-154 cells were infected in GM3 with different doses (indicated as the dilution fold above the brackets) of stock adenoviruses expressing control (C) or HA-CA-ALK5 (A) for 5 h. Whole cell lysates were collected after an additional 29 h and analyzed by Western blot.

    Techniques Used: Activity Assay, Expressing, Western Blot, Transfection, Luciferase, Construct, Infection

    TGF-β represses survivin promoter within the region −182/−4, and the CDE/CHR elements within this region are essential for this TGF-β’s effect (A) Left: Diagram of survivin promoter 5’-deletion constructs. Right: Individual promoter constructs (0.4 µg) were transfected into NRP-154 cells and treated with or without TGF-β (10 ng/ml) for 25 h. (B) Schematic representation of the nucleotide changes in survivin promoter mutants, which were generated with Suv-370 reporter plasmid. SBE2 and AP-2 sites are underlined, while CDE and CHR sites are boxed. All mutated nucleotides are indicated in inverted display. Note that CDE(m) disrupts both AP-2 and CDE sites. Although not shown here, SBE1(m) has the same nucleotide changes as SBE2(m). (C, D, E) Suv-370 wild type (WT) or mutant promoter reporter plasmid (0.4 µg) was transfected into NRP-154 cells and treated with 10 ng/ml of TGF-β1 or vehicle for 24 h. Suppression for each promoter construct was calculated as %Suppression = [(L V -L T )/L V ] * 100% (L: average luciferase activity; V: vehicle; T: TGF-β). The luciferase activity of the first bar was arbitrarily set as 1. Note that data in (D) and (E) are from the same experiment, and the negative value of % suppression in (E) reflects enhancement.
    Figure Legend Snippet: TGF-β represses survivin promoter within the region −182/−4, and the CDE/CHR elements within this region are essential for this TGF-β’s effect (A) Left: Diagram of survivin promoter 5’-deletion constructs. Right: Individual promoter constructs (0.4 µg) were transfected into NRP-154 cells and treated with or without TGF-β (10 ng/ml) for 25 h. (B) Schematic representation of the nucleotide changes in survivin promoter mutants, which were generated with Suv-370 reporter plasmid. SBE2 and AP-2 sites are underlined, while CDE and CHR sites are boxed. All mutated nucleotides are indicated in inverted display. Note that CDE(m) disrupts both AP-2 and CDE sites. Although not shown here, SBE1(m) has the same nucleotide changes as SBE2(m). (C, D, E) Suv-370 wild type (WT) or mutant promoter reporter plasmid (0.4 µg) was transfected into NRP-154 cells and treated with 10 ng/ml of TGF-β1 or vehicle for 24 h. Suppression for each promoter construct was calculated as %Suppression = [(L V -L T )/L V ] * 100% (L: average luciferase activity; V: vehicle; T: TGF-β). The luciferase activity of the first bar was arbitrarily set as 1. Note that data in (D) and (E) are from the same experiment, and the negative value of % suppression in (E) reflects enhancement.

    Techniques Used: Construct, Transfection, Generated, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay

    Regulation of survivin by TGF-β in prostate cells correlate with their malignancy (A) TGF-β time course in NRP-152 cells. (B) Human prostate cancer cell lines DU-145 and PC-3 plated in DMEM/F12 + 1% FBS medium, and rat prostate epithelial cell lines NRP-154 (tumorigenic) and NRP-152 (non-tumorigenic) plated in GM3 medium were treated with vehicle or 10 ng/ml of TGF-β1 for 24 h, and cell lysates were subjected to Western blot analysis. Lower exp: lower exposure. (C) Non-tumorigenic human prostate cell line RWPE-1 was treated with vehicle or 10 ng/ml of TGF-β1 for the indicated time. (D) LNCaP and C4-2B cells were plated in 1% DC-FBS+15 mM HEPES+DMEM/F12, infected with adenovirus expressing TβRII (1/200 dilution), and treated with 20 ng/ml of epidermal growth factor. Cells were then treated with or without 10 nM of DHT for 24 h, followed by the incubation with vehicle or 10 ng/ml of TGF-β1 for an additional 24 h. (E) A schematic model of survivin regulation by TGF-β and its implication in prostate cancer. TGF-β ligand-bound receptors activate Smads 2 and 3, which activate Rb (and/or Rb-like proteins) by inducing its hypophosphorylation. Activated Rb can then bind E2F4 to form a repressor complex on the survivin promoter through CDE/CHR elements. Smad3, which binds to SBE upstream of CDE/CHR in vitro , may also contribute to TGF-β-mediated survivin promoter suppression through interaction with E2F4. In normal cells, the TGF-β/survivin regulatory axis is usually intact to maintain survivin levels. However, this regulatory axis is often defective in malignant cancer cells, resulting in heightened levels of survivin and thus resistance to chemotherapeutics.
    Figure Legend Snippet: Regulation of survivin by TGF-β in prostate cells correlate with their malignancy (A) TGF-β time course in NRP-152 cells. (B) Human prostate cancer cell lines DU-145 and PC-3 plated in DMEM/F12 + 1% FBS medium, and rat prostate epithelial cell lines NRP-154 (tumorigenic) and NRP-152 (non-tumorigenic) plated in GM3 medium were treated with vehicle or 10 ng/ml of TGF-β1 for 24 h, and cell lysates were subjected to Western blot analysis. Lower exp: lower exposure. (C) Non-tumorigenic human prostate cell line RWPE-1 was treated with vehicle or 10 ng/ml of TGF-β1 for the indicated time. (D) LNCaP and C4-2B cells were plated in 1% DC-FBS+15 mM HEPES+DMEM/F12, infected with adenovirus expressing TβRII (1/200 dilution), and treated with 20 ng/ml of epidermal growth factor. Cells were then treated with or without 10 nM of DHT for 24 h, followed by the incubation with vehicle or 10 ng/ml of TGF-β1 for an additional 24 h. (E) A schematic model of survivin regulation by TGF-β and its implication in prostate cancer. TGF-β ligand-bound receptors activate Smads 2 and 3, which activate Rb (and/or Rb-like proteins) by inducing its hypophosphorylation. Activated Rb can then bind E2F4 to form a repressor complex on the survivin promoter through CDE/CHR elements. Smad3, which binds to SBE upstream of CDE/CHR in vitro , may also contribute to TGF-β-mediated survivin promoter suppression through interaction with E2F4. In normal cells, the TGF-β/survivin regulatory axis is usually intact to maintain survivin levels. However, this regulatory axis is often defective in malignant cancer cells, resulting in heightened levels of survivin and thus resistance to chemotherapeutics.

    Techniques Used: Western Blot, Infection, Expressing, Incubation, In Vitro

    12) Product Images from "X-linked Inhibitor of Apoptosis Protein and Its E3 Ligase Activity Promote Transforming Growth Factor-?-mediated Nuclear Factor-?B Activation during Breast Cancer Progression *"

    Article Title: X-linked Inhibitor of Apoptosis Protein and Its E3 Ligase Activity Promote Transforming Growth Factor-?-mediated Nuclear Factor-?B Activation during Breast Cancer Progression *

    Journal:

    doi: 10.1074/jbc.M109.018374

    xIAP-deficient MEFs exhibit reduced TGF-β signaling. A , WT and xIAP-deficient ( xIAP −/− ) MEFs were transiently transfected with pSBE-luciferase and β-galactosidase followed by overnight stimulation with TGF-β1 (5
    Figure Legend Snippet: xIAP-deficient MEFs exhibit reduced TGF-β signaling. A , WT and xIAP-deficient ( xIAP −/− ) MEFs were transiently transfected with pSBE-luciferase and β-galactosidase followed by overnight stimulation with TGF-β1 (5

    Techniques Used: Transfection, Luciferase

    13) Product Images from "A Local Paracrine and Endocrine Network Involving TGF?, Cox-2, ROS, and Estrogen Receptor ? Influences Reactive Stromal Cell Regulation of Prostate Cancer Cell Motility"

    Article Title: A Local Paracrine and Endocrine Network Involving TGF?, Cox-2, ROS, and Estrogen Receptor ? Influences Reactive Stromal Cell Regulation of Prostate Cancer Cell Motility

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2011-1371

    TGF-β1 treatment triggers H 2 O 2 production in reactive stromal cells, which can limit SMIF activity. A, In response to exogenous TGF-β1, WPMY-1 cells produce increased levels of H 2 O 2 as measured by an endpoint Amplex Red assay. WPMY-1 cells
    Figure Legend Snippet: TGF-β1 treatment triggers H 2 O 2 production in reactive stromal cells, which can limit SMIF activity. A, In response to exogenous TGF-β1, WPMY-1 cells produce increased levels of H 2 O 2 as measured by an endpoint Amplex Red assay. WPMY-1 cells

    Techniques Used: Activity Assay, Amplex Red Assay

    14) Product Images from "p53 induces miR199a-3p to suppress SOCS7 for STAT3 activation and renal fibrosis in UUO"

    Article Title: p53 induces miR199a-3p to suppress SOCS7 for STAT3 activation and renal fibrosis in UUO

    Journal: Scientific Reports

    doi: 10.1038/srep43409

    Pifithrin-α suppressed TGF-β1-induced STAT3 activation and miR-199a-3p expression. Cultured HK-2 cells were treated with 10 ng/ml TGF-β1 or 10 μM pifithrin-a for 0 to 24 h or transfection of miR-199a-3p or negative control analog or SOCS7 siRNA, followed by immunoblot for p-STAT3, STAT3, and ECM genes, and real time PCR for miR-199a-3p. Immunoblot ( A ) analysis of p-p53 Ser 15 and p53, densitometry ( B ) of proteins signals on immunoblots, and real time PCR analysis ( C ) of miR-199a-3p after indicated time point of treatment with TGF-β1. Relative protein levels ( D ) of vimentin, COL1, and β-actin 24 h after transfection of miR-199a-3p analog (100 nM) or miR analog negative control (miR-ANC) with or without TGF-β1 treatment, densitometry ( E ) of proteins signals on immunoblots. Immunoblot analysis ( F ) of vimentin, COL1, SOCS7, p-STAT3, STAT3 and β-actin, densitometry ( G ) of proteins signals on immunoblots, and real time PCR analysis ( H ) of miR-199a-3p 24 h after TGF-β1 alone or TGF-β1 plus pifithrin-a treatment. Data were expressed as means ± sd (n = 6); # p
    Figure Legend Snippet: Pifithrin-α suppressed TGF-β1-induced STAT3 activation and miR-199a-3p expression. Cultured HK-2 cells were treated with 10 ng/ml TGF-β1 or 10 μM pifithrin-a for 0 to 24 h or transfection of miR-199a-3p or negative control analog or SOCS7 siRNA, followed by immunoblot for p-STAT3, STAT3, and ECM genes, and real time PCR for miR-199a-3p. Immunoblot ( A ) analysis of p-p53 Ser 15 and p53, densitometry ( B ) of proteins signals on immunoblots, and real time PCR analysis ( C ) of miR-199a-3p after indicated time point of treatment with TGF-β1. Relative protein levels ( D ) of vimentin, COL1, and β-actin 24 h after transfection of miR-199a-3p analog (100 nM) or miR analog negative control (miR-ANC) with or without TGF-β1 treatment, densitometry ( E ) of proteins signals on immunoblots. Immunoblot analysis ( F ) of vimentin, COL1, SOCS7, p-STAT3, STAT3 and β-actin, densitometry ( G ) of proteins signals on immunoblots, and real time PCR analysis ( H ) of miR-199a-3p 24 h after TGF-β1 alone or TGF-β1 plus pifithrin-a treatment. Data were expressed as means ± sd (n = 6); # p

    Techniques Used: Activation Assay, Expressing, Cell Culture, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot

    MiR-199a-3p suppressed SOCS7 to increase STAT3 activation. Cultured HK-2 cells were treated with transfection of miR-199a-3p or negative control analog or SOCS7 siRNA, followed by immunoblot for p-STAT3, STAT3, and ECM genes, and immunoprecipitation with antibodies to p53. ( A ) Relative protein levels of SOCS7 and β-actin 24 hours after transfection of miR-199a-3p analog (100 nM) or miR-ANC, densitometry ( B ) of proteins signals on immunoblots. ( C ) Detected luciferase activity 24 hours after cotransfection of miR-199a-3p analog (100 nM) or miR-ANC with SOCS7 3 ’UTR luciferase reporter vector. ( D ) Relative protein levels of p-STAT3 (Tyr705) and STAT3 24 hours after the transfection of SOCS7 siRNA or siRNA-NC, densitometry ( E ) of proteins signals on immunoblots. ( F ) ChIP assays for p53 were performed with chromatin material isolated from HK2 cells treated with TGF-β1. Precipitated DNA was amplied with oligonucleotides spanning regions of the potential p53 binding sites1 and 2 (pBS1and pBS2); total inputs were indicated. The antibody against p53 immunoprecipitated the DNA fragments from HK2 cells containing the potential pBS1and pBS2. Data were expressed as means ± sd (n = 6); # p
    Figure Legend Snippet: MiR-199a-3p suppressed SOCS7 to increase STAT3 activation. Cultured HK-2 cells were treated with transfection of miR-199a-3p or negative control analog or SOCS7 siRNA, followed by immunoblot for p-STAT3, STAT3, and ECM genes, and immunoprecipitation with antibodies to p53. ( A ) Relative protein levels of SOCS7 and β-actin 24 hours after transfection of miR-199a-3p analog (100 nM) or miR-ANC, densitometry ( B ) of proteins signals on immunoblots. ( C ) Detected luciferase activity 24 hours after cotransfection of miR-199a-3p analog (100 nM) or miR-ANC with SOCS7 3 ’UTR luciferase reporter vector. ( D ) Relative protein levels of p-STAT3 (Tyr705) and STAT3 24 hours after the transfection of SOCS7 siRNA or siRNA-NC, densitometry ( E ) of proteins signals on immunoblots. ( F ) ChIP assays for p53 were performed with chromatin material isolated from HK2 cells treated with TGF-β1. Precipitated DNA was amplied with oligonucleotides spanning regions of the potential p53 binding sites1 and 2 (pBS1and pBS2); total inputs were indicated. The antibody against p53 immunoprecipitated the DNA fragments from HK2 cells containing the potential pBS1and pBS2. Data were expressed as means ± sd (n = 6); # p

    Techniques Used: Activation Assay, Cell Culture, Transfection, Negative Control, Immunoprecipitation, Western Blot, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Chromatin Immunoprecipitation, Isolation, Binding Assay

    The role and molecular mechanism of p53 in UUO-induced renal fibrosis. TGF-β1 increased miR-199a-3p expression by induction of p53. Furthermore, miR-199a-3p directly suppressed SOCS7 expression, which led to activation of STAT3 and upregulation of the production of probrotic proteins.
    Figure Legend Snippet: The role and molecular mechanism of p53 in UUO-induced renal fibrosis. TGF-β1 increased miR-199a-3p expression by induction of p53. Furthermore, miR-199a-3p directly suppressed SOCS7 expression, which led to activation of STAT3 and upregulation of the production of probrotic proteins.

    Techniques Used: Expressing, Activation Assay

    15) Product Images from "Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells"

    Article Title: Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0572-7

    Resistance to EGFR inhibitors in mesenchymal T-Epi cells. T-Epi cells were treated with recombinant TGF-β1 (5 ng/mL) for 14 days to establish mesenchymal T-Epi cells. Mesenchymal T-Epi cells were cultured with normal KSFM medium (TGF-β1 free) for 24 h, and then parental T-Epi and mesenchymal T-Epi cells were treated with erlotinib or cetuximab. a Phase-contrast images of parental T-Epi cells and mesenchymal T-Epi cells. Note that T-Epi cells treated with TGF-β1 changed to spindle-shaped cells. b Protein levels of E-cadherin and vimentin in parental T-Epi and mesenchymal T-Epi cells, determined by western blotting. c Phosphorylated- and total-EGFR protein levels in parental T-Epi and mesenchymal T-Epi cells treated with erlotinib or cetuximab, determined by western blotting. The inhibitory effect of EGFR phosphorylation due to EGFR inhibitors was lower in mesenchymal T-Epi cells compared with parental T-Epi cells. d Cell growth of mesenchymal T-Epi cells treated with or without erlotinib or cetuximab. The results are presented as means ± SD (bars). (n.s., not significant, vs vehicle control; n = 3) Inhibition of cell growth was not observed in mesenchymal T-Epi cells. e Involucrin protein levels in parental T-Epi and mesenchymal T-Epi cells treated with or without erlotinib or cetuximab, determined by western blotting. The promotion of involucrin expression due to treatment with EGFR inhibitors was more suppressed in mesenchymal T-Epi cells compared with parental T-Epi cells
    Figure Legend Snippet: Resistance to EGFR inhibitors in mesenchymal T-Epi cells. T-Epi cells were treated with recombinant TGF-β1 (5 ng/mL) for 14 days to establish mesenchymal T-Epi cells. Mesenchymal T-Epi cells were cultured with normal KSFM medium (TGF-β1 free) for 24 h, and then parental T-Epi and mesenchymal T-Epi cells were treated with erlotinib or cetuximab. a Phase-contrast images of parental T-Epi cells and mesenchymal T-Epi cells. Note that T-Epi cells treated with TGF-β1 changed to spindle-shaped cells. b Protein levels of E-cadherin and vimentin in parental T-Epi and mesenchymal T-Epi cells, determined by western blotting. c Phosphorylated- and total-EGFR protein levels in parental T-Epi and mesenchymal T-Epi cells treated with erlotinib or cetuximab, determined by western blotting. The inhibitory effect of EGFR phosphorylation due to EGFR inhibitors was lower in mesenchymal T-Epi cells compared with parental T-Epi cells. d Cell growth of mesenchymal T-Epi cells treated with or without erlotinib or cetuximab. The results are presented as means ± SD (bars). (n.s., not significant, vs vehicle control; n = 3) Inhibition of cell growth was not observed in mesenchymal T-Epi cells. e Involucrin protein levels in parental T-Epi and mesenchymal T-Epi cells treated with or without erlotinib or cetuximab, determined by western blotting. The promotion of involucrin expression due to treatment with EGFR inhibitors was more suppressed in mesenchymal T-Epi cells compared with parental T-Epi cells

    Techniques Used: Recombinant, Cell Culture, Western Blot, Inhibition, Expressing

    16) Product Images from "Tri-methylation of H3K79 is decreased in TGF-β1-induced epithelial-to-mesenchymal transition in lung cancer"

    Article Title: Tri-methylation of H3K79 is decreased in TGF-β1-induced epithelial-to-mesenchymal transition in lung cancer

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-017-0380-0

    Partial reversion of TGF-β1-induced EMT by epigenetic inhibitors in H358 cells. a Protocol: Cells were treated simultaneously with TGF-β1 (10 ng/ml), EPZ5676 (1 μM), SGC0946 (5 μM), or PFI-1 (5 μM) for 48 h and with SAHA (5 μM) for the last 12 h. b mRNA expression was measured by RT-qPCR and normalized to GAPDH mRNA for control cells (white bars) and TGF-β1 treated cells (black bars). The graph corresponds to the mean ± SD of two independent experiments with PCR in duplicate. * p
    Figure Legend Snippet: Partial reversion of TGF-β1-induced EMT by epigenetic inhibitors in H358 cells. a Protocol: Cells were treated simultaneously with TGF-β1 (10 ng/ml), EPZ5676 (1 μM), SGC0946 (5 μM), or PFI-1 (5 μM) for 48 h and with SAHA (5 μM) for the last 12 h. b mRNA expression was measured by RT-qPCR and normalized to GAPDH mRNA for control cells (white bars) and TGF-β1 treated cells (black bars). The graph corresponds to the mean ± SD of two independent experiments with PCR in duplicate. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    TGF-β1 induces EMT in NSCLC cell lines. Expression of epithelial and mesenchymal markers was determined by mRNA and protein analysis of A549 (white bars) and H358 (black bars) cells cultured with TGF-β1 (10 ng/ml) for the indicated time (h). a mRNA expression was measured by RT-qPCR and normalized to GAPDH mRNA level. The graph corresponds to the mean ± SD of two independent experiments with PCR in duplicate. * p
    Figure Legend Snippet: TGF-β1 induces EMT in NSCLC cell lines. Expression of epithelial and mesenchymal markers was determined by mRNA and protein analysis of A549 (white bars) and H358 (black bars) cells cultured with TGF-β1 (10 ng/ml) for the indicated time (h). a mRNA expression was measured by RT-qPCR and normalized to GAPDH mRNA level. The graph corresponds to the mean ± SD of two independent experiments with PCR in duplicate. * p

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Polymerase Chain Reaction

    TGF-β1-induced EMT increases PD-L1 expression in H358 cells. Cells were treated with TGF-β1 (10 ng/ml) for 48 h. a PD-L1 mRNA expression was measured by RT-qPCR and normalized to GAPDH mRNA. The graph corresponds to the mean ± SD of three independent experiments. *** p
    Figure Legend Snippet: TGF-β1-induced EMT increases PD-L1 expression in H358 cells. Cells were treated with TGF-β1 (10 ng/ml) for 48 h. a PD-L1 mRNA expression was measured by RT-qPCR and normalized to GAPDH mRNA. The graph corresponds to the mean ± SD of three independent experiments. *** p

    Techniques Used: Expressing, Quantitative RT-PCR

    TGF-β1-induced EMT represses DOT1L in H358 cells. Cells were treated with TGF-β1 (10 ng/ml) for 48 h. a DOT1L mRNA expression was measured by RT-qPCR and normalized to GAPDH mRNA. The graph corresponds to the mean ± SD of three independent experiments with PCR in duplicate. ** p = 0.0056 by Student’s t test. b DOT1L protein was analyzed by immunoblotting of A549 and H358 total cell lysates. Actin was used as a loading control. The apparent molecular weights (kDa) are indicated on the right of the panel. This blot is representative of four independent experiments. c The graph corresponds to the quantification of the intensity of the protein bands for A549 and H358 cell lines with (white bars) or without TGF-β1 (black bars) treatment after normalization to actin for DOT1L and total histone H3 for H3K79me3. Value 1 corresponds to untreated cells. Statistical analysis was performed for four independent experiments with the Student’s t test (** corresponds to p
    Figure Legend Snippet: TGF-β1-induced EMT represses DOT1L in H358 cells. Cells were treated with TGF-β1 (10 ng/ml) for 48 h. a DOT1L mRNA expression was measured by RT-qPCR and normalized to GAPDH mRNA. The graph corresponds to the mean ± SD of three independent experiments with PCR in duplicate. ** p = 0.0056 by Student’s t test. b DOT1L protein was analyzed by immunoblotting of A549 and H358 total cell lysates. Actin was used as a loading control. The apparent molecular weights (kDa) are indicated on the right of the panel. This blot is representative of four independent experiments. c The graph corresponds to the quantification of the intensity of the protein bands for A549 and H358 cell lines with (white bars) or without TGF-β1 (black bars) treatment after normalization to actin for DOT1L and total histone H3 for H3K79me3. Value 1 corresponds to untreated cells. Statistical analysis was performed for four independent experiments with the Student’s t test (** corresponds to p

    Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    TGF-β1-induced EMT promotes histone post-translational modifications. A549 and H358 cells were treated with TGF-β1 (10 ng/ml) for the indicated time. a Histone marks were detected by immunoblotting from total cell lysates. H3 total (H3T) was used as a loading control. The apparent molecular weights (kDa) are indicated on the right of the panel. The intensity of the protein bands was quantified after normalization to H3T and compared to control cells (0 h) standardized to one. b H3K79me3 (green) was detected by confocal microscopy. DNA (red) was stained with DRAQ5
    Figure Legend Snippet: TGF-β1-induced EMT promotes histone post-translational modifications. A549 and H358 cells were treated with TGF-β1 (10 ng/ml) for the indicated time. a Histone marks were detected by immunoblotting from total cell lysates. H3 total (H3T) was used as a loading control. The apparent molecular weights (kDa) are indicated on the right of the panel. The intensity of the protein bands was quantified after normalization to H3T and compared to control cells (0 h) standardized to one. b H3K79me3 (green) was detected by confocal microscopy. DNA (red) was stained with DRAQ5

    Techniques Used: Confocal Microscopy, Staining

    17) Product Images from "Transforming growth factor-beta 1 (TGF-?1) expression in ovine lentivirus-induced lymphoid interstitial pneumonia"

    Article Title: Transforming growth factor-beta 1 (TGF-?1) expression in ovine lentivirus-induced lymphoid interstitial pneumonia

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.1998.00553.x

    TGF-β1 staining in lung sections from seropositive sheep with grade II (a) and grade III (b) histopathological lesions. Strong staining was observed in central area of lymphoid follicle (a). (Original mag. × 100.) In contrast, weak reactivity was observed in large lymphoid follicle (b). (Original mag. × 400.) In (b), three high endothelial venules with strong staining located characteristically in the periphery of the follicle are shown (arrows).
    Figure Legend Snippet: TGF-β1 staining in lung sections from seropositive sheep with grade II (a) and grade III (b) histopathological lesions. Strong staining was observed in central area of lymphoid follicle (a). (Original mag. × 100.) In contrast, weak reactivity was observed in large lymphoid follicle (b). (Original mag. × 400.) In (b), three high endothelial venules with strong staining located characteristically in the periphery of the follicle are shown (arrows).

    Techniques Used: Staining

    TGF-β1 staining in lung sections from seropositive sheep with grade II histopathological lesions. Strong TGF-β1 staining was sometimes observed in interstitial venules with hypertrophied endothelium (arrows). These were always located in areas with interstitial inflammatory reactions. (Original mag. × 400.)
    Figure Legend Snippet: TGF-β1 staining in lung sections from seropositive sheep with grade II histopathological lesions. Strong TGF-β1 staining was sometimes observed in interstitial venules with hypertrophied endothelium (arrows). These were always located in areas with interstitial inflammatory reactions. (Original mag. × 400.)

    Techniques Used: Staining

    TGF-β1 staining in lung section from seropositive sheep with grade III histopathological lesions. Numerous inflammatory cells with weak or no TGF-β1 staining were observed close to hyperplastic smooth muscle. Loose aggregates of macrophages also showed weak TGF-β1 immunostaining (arrows). (Original mag. × 400.)
    Figure Legend Snippet: TGF-β1 staining in lung section from seropositive sheep with grade III histopathological lesions. Numerous inflammatory cells with weak or no TGF-β1 staining were observed close to hyperplastic smooth muscle. Loose aggregates of macrophages also showed weak TGF-β1 immunostaining (arrows). (Original mag. × 400.)

    Techniques Used: Staining, Immunostaining

    TGF-β1 staining in normal lung
    Figure Legend Snippet: TGF-β1 staining in normal lung

    Techniques Used: Staining

    TGF-β1 staining in control sheep. (a) Non-fixation fixed lung. Airway showing strong TGF-β1 staining in epithelium (× 100). (b) Non-fixation fixed lung. Small blood vessel with TGF-β1 immunostaining in endothelial cells and lumen but not in smooth muscle cells. (Original mag. × 400.) (c) Fixation fixed lung. Alveolar macrophage showing strong TGF-β1 staining. No reactivity is observed in interstitial cells and stroma. (Original mag. × 400.)
    Figure Legend Snippet: TGF-β1 staining in control sheep. (a) Non-fixation fixed lung. Airway showing strong TGF-β1 staining in epithelium (× 100). (b) Non-fixation fixed lung. Small blood vessel with TGF-β1 immunostaining in endothelial cells and lumen but not in smooth muscle cells. (Original mag. × 400.) (c) Fixation fixed lung. Alveolar macrophage showing strong TGF-β1 staining. No reactivity is observed in interstitial cells and stroma. (Original mag. × 400.)

    Techniques Used: Staining, Immunostaining

    TGF-β1 staining in normal lung
    Figure Legend Snippet: TGF-β1 staining in normal lung

    Techniques Used: Staining

    TGF-β1 staining in lung sections from seropositive sheep with grade I histopathological lesions. (a) Non-fixation fixed lung. Increasing TGF-β1 staining in macrophages. (b) Fixation fixed lung. Increasing TGF-β1 staining in stroma (original mag. × 400).
    Figure Legend Snippet: TGF-β1 staining in lung sections from seropositive sheep with grade I histopathological lesions. (a) Non-fixation fixed lung. Increasing TGF-β1 staining in macrophages. (b) Fixation fixed lung. Increasing TGF-β1 staining in stroma (original mag. × 400).

    Techniques Used: Staining

    18) Product Images from "Zyxin Mediates Actin Fiber Reorganization in Epithelial-Mesenchymal Transition and Contributes to Endocardial Morphogenesis"

    Article Title: Zyxin Mediates Actin Fiber Reorganization in Epithelial-Mesenchymal Transition and Contributes to Endocardial Morphogenesis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E09-01-0046

    Zyxin is up-regulated and relocates to actin stress fibers in TGF-β1–induced EMT. (A–D) TGF-β1–induced in vitro EMT in NMuMG-C7 cells. NMuMG-C7 cells treated with TGF-β1 for 24 h exhibit the cell scattering (A), actin stress fiber formation (B), down-regulation of E-cadherin (C), and up-regulation of N-cadherin (D). Scale bars, 100 μm. (E) Real-time PCR analysis of zyxin mRNA in NMuMG-C7 cells treated with TGF-β1. Results are shown as the mean ± SEM (n = 3). (F) Immunoblot analysis of zyxin in NMuMG-C7 cells treated with TGF-β1. GAPDH was used as a loading control. (G) TGF-β1 induced zyxin relocation from focal adhesions to actin fibers. Confocal microscopy of EGFP-tagged zyxin (green) in NMuMG-C7 cells treated or not with TGF-β1 for 24 h. Zyxin locates at focal adhesions before TGF-β1 treatment and mobilizes to actin fibers after the treatment (Also see Supplementary Video 1). The cells were counterstained with phalloidin (red) and DAPI (blue). Scale bars, 50 μm.
    Figure Legend Snippet: Zyxin is up-regulated and relocates to actin stress fibers in TGF-β1–induced EMT. (A–D) TGF-β1–induced in vitro EMT in NMuMG-C7 cells. NMuMG-C7 cells treated with TGF-β1 for 24 h exhibit the cell scattering (A), actin stress fiber formation (B), down-regulation of E-cadherin (C), and up-regulation of N-cadherin (D). Scale bars, 100 μm. (E) Real-time PCR analysis of zyxin mRNA in NMuMG-C7 cells treated with TGF-β1. Results are shown as the mean ± SEM (n = 3). (F) Immunoblot analysis of zyxin in NMuMG-C7 cells treated with TGF-β1. GAPDH was used as a loading control. (G) TGF-β1 induced zyxin relocation from focal adhesions to actin fibers. Confocal microscopy of EGFP-tagged zyxin (green) in NMuMG-C7 cells treated or not with TGF-β1 for 24 h. Zyxin locates at focal adhesions before TGF-β1 treatment and mobilizes to actin fibers after the treatment (Also see Supplementary Video 1). The cells were counterstained with phalloidin (red) and DAPI (blue). Scale bars, 50 μm.

    Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Confocal Microscopy

    Twist1 regulates zyxin in TGF-β1-EMT. (A) Expression analysis of EMT inducers in NMuMG-C7 cells. RT-PCR performed with total RNA isolated from NMuMG-C7 cells treated with or without TGF-β1. β-Actin was used as a control. (B) Twist1 up-regulates zyxin. Real-time PCR performed with total RNA from NMuMG-C7 cells transduced with EGFP, Snail, Twist1, or HMGA2. *p = 0.0122 versus EGFP. Error bars, the SEM of three technical replicates. (C) Twist1 induced the expression of zyxin. Immunoblot analysis of zyxin in NMuMG-C7 cells transduced with EGFP, EGFP-Snail, EGFP-Twist1, or HMGA2. The expression of transduced genes was confirmed by blotting with anti-EGFP and anti-HMGA2 antibodies. (D) Efficacy of Twist1 RNAi assessed by RT-PCR. Total RNA from NMuMG-C7 cells transfected with either Twist1 siRNA or scramble RNA were subjected to RT-PCR. (E) Twist1 depletion abrogated up-regulation of zyxin by TGF-β1. Real-time PCR analysis of zyxin mRNA in NMuMG-C7 cells transfected with Twist1 siRNA or scramble RNA and treated or not with TGF-β1. Results are shown as the mean ± SEM (n = 3). *p = 0.011 when compared with control, TGF-β1(−); **p = 0.0236 when compared with control, TGF-β1(+). (F) Twist1 depletion suppressed the expression of zyxin. Immunoblot analysis of zyxin in NMuMG-C7 cells transfected with Twist1 siRNA or scramble RNA and treated or not with TGF-β1. GAPDH was used as a loading control. (G) Results of F were quantitated by densitometry using NIH ImageJ. Error bars, SD from triplicate experiments. *p = 0.0084 when compared with control, TGF-β1(−); **p = 0.035 when compared with control, TGF-β1(−); ***p = 0.012 when compared with control, TGF-β1(+).
    Figure Legend Snippet: Twist1 regulates zyxin in TGF-β1-EMT. (A) Expression analysis of EMT inducers in NMuMG-C7 cells. RT-PCR performed with total RNA isolated from NMuMG-C7 cells treated with or without TGF-β1. β-Actin was used as a control. (B) Twist1 up-regulates zyxin. Real-time PCR performed with total RNA from NMuMG-C7 cells transduced with EGFP, Snail, Twist1, or HMGA2. *p = 0.0122 versus EGFP. Error bars, the SEM of three technical replicates. (C) Twist1 induced the expression of zyxin. Immunoblot analysis of zyxin in NMuMG-C7 cells transduced with EGFP, EGFP-Snail, EGFP-Twist1, or HMGA2. The expression of transduced genes was confirmed by blotting with anti-EGFP and anti-HMGA2 antibodies. (D) Efficacy of Twist1 RNAi assessed by RT-PCR. Total RNA from NMuMG-C7 cells transfected with either Twist1 siRNA or scramble RNA were subjected to RT-PCR. (E) Twist1 depletion abrogated up-regulation of zyxin by TGF-β1. Real-time PCR analysis of zyxin mRNA in NMuMG-C7 cells transfected with Twist1 siRNA or scramble RNA and treated or not with TGF-β1. Results are shown as the mean ± SEM (n = 3). *p = 0.011 when compared with control, TGF-β1(−); **p = 0.0236 when compared with control, TGF-β1(+). (F) Twist1 depletion suppressed the expression of zyxin. Immunoblot analysis of zyxin in NMuMG-C7 cells transfected with Twist1 siRNA or scramble RNA and treated or not with TGF-β1. GAPDH was used as a loading control. (G) Results of F were quantitated by densitometry using NIH ImageJ. Error bars, SD from triplicate experiments. *p = 0.0084 when compared with control, TGF-β1(−); **p = 0.035 when compared with control, TGF-β1(−); ***p = 0.012 when compared with control, TGF-β1(+).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction, Transduction, Transfection

    Zyxin is essential for cell migration and actin fiber reorganization in TGF-β1–induced EMT. (A) Immunoblot analysis of zyxin and E-and N-cadherin in NMuMG-C7 cells transfected with zyxin siRNA or scramble RNA. After treatment with TGF-β1 for 24 h, total lysates were subjected to Western blot. GAPDH was used as a loading control. (B) Zyxin depletion abolished cell motility in TGF-β1-EMT. Cell motility was assessed by modified Boyden's chamber assay. NMuMG-C7 cells were transfected with zyxin siRNA or scramble RNA and treated or not with TGF-β1 for 24 h. The cells that migrated to the lower surface of the membrane were stained after 4 h of incubation. Scale bar, 250 μm. (C) The number of migrated cells in B. *p = 0.0344 versus Scramble RNA, TGF-β1 (−); **p = 0.0159 versus Scramble RNA, TGF-β1 (+). Results are shown as the mean ± SEM of four separate migration assays from two independent experiments. (D–K) Zyxin is essential for actin fiber formation in TGF-β1-EMT. Phalloidin staining was performed with NMuMG-C7 cells transfected with zyxin siRNA or scramble RNA and treated or not with TGF-β1 for 24 h. Apical and basal actin network were observed separately with a confocal microscope. Note that both the apical and basal actin fiber formation were hampered by zyxin depletion. Scale bar, 50 μm. (L) The scheme of EGFP-tagged zyxin deletion constructs. EGFP–zyxin constructs were introduced into NMuMG-C7 cells by transient transfection. FL: full length. (M) NMuMG-C7 cells were transduced with lentivirus vector that expresses shZyxin that targets 3′UTR. The resultant cells were then transfected with the EGFP–zyxin constructs that lack 3′UTR. The suppression of endogenous zyxin and the expression of transfected genes were confirmed by blotting with anti-zyxin and ant-EGFP antibodies. LIM only construct lacks the epitope recognized by zyxin antibody. (N) Neither ΔLIM nor LIM only constructs rescued the endogenous zyxin depletion. Endogenous zyxin-depleted cells were transfected with EGFP–zyxin constructs and treated with TGF-β1 for 24 h. Actin fiber formation was observed in the cells transfected with FL (arrowhead), but not with ΔLIM or LIM only constructs. Scale bar, 50 μm.
    Figure Legend Snippet: Zyxin is essential for cell migration and actin fiber reorganization in TGF-β1–induced EMT. (A) Immunoblot analysis of zyxin and E-and N-cadherin in NMuMG-C7 cells transfected with zyxin siRNA or scramble RNA. After treatment with TGF-β1 for 24 h, total lysates were subjected to Western blot. GAPDH was used as a loading control. (B) Zyxin depletion abolished cell motility in TGF-β1-EMT. Cell motility was assessed by modified Boyden's chamber assay. NMuMG-C7 cells were transfected with zyxin siRNA or scramble RNA and treated or not with TGF-β1 for 24 h. The cells that migrated to the lower surface of the membrane were stained after 4 h of incubation. Scale bar, 250 μm. (C) The number of migrated cells in B. *p = 0.0344 versus Scramble RNA, TGF-β1 (−); **p = 0.0159 versus Scramble RNA, TGF-β1 (+). Results are shown as the mean ± SEM of four separate migration assays from two independent experiments. (D–K) Zyxin is essential for actin fiber formation in TGF-β1-EMT. Phalloidin staining was performed with NMuMG-C7 cells transfected with zyxin siRNA or scramble RNA and treated or not with TGF-β1 for 24 h. Apical and basal actin network were observed separately with a confocal microscope. Note that both the apical and basal actin fiber formation were hampered by zyxin depletion. Scale bar, 50 μm. (L) The scheme of EGFP-tagged zyxin deletion constructs. EGFP–zyxin constructs were introduced into NMuMG-C7 cells by transient transfection. FL: full length. (M) NMuMG-C7 cells were transduced with lentivirus vector that expresses shZyxin that targets 3′UTR. The resultant cells were then transfected with the EGFP–zyxin constructs that lack 3′UTR. The suppression of endogenous zyxin and the expression of transfected genes were confirmed by blotting with anti-zyxin and ant-EGFP antibodies. LIM only construct lacks the epitope recognized by zyxin antibody. (N) Neither ΔLIM nor LIM only constructs rescued the endogenous zyxin depletion. Endogenous zyxin-depleted cells were transfected with EGFP–zyxin constructs and treated with TGF-β1 for 24 h. Actin fiber formation was observed in the cells transfected with FL (arrowhead), but not with ΔLIM or LIM only constructs. Scale bar, 50 μm.

    Techniques Used: Migration, Transfection, Western Blot, Modification, Boyden Chamber Assay, Staining, Incubation, Microscopy, Construct, Transduction, Plasmid Preparation, Expressing

    19) Product Images from "A subset of IL-17+ mesenchymal stem cells possesses anti-Candida albicans effect"

    Article Title: A subset of IL-17+ mesenchymal stem cells possesses anti-Candida albicans effect

    Journal: Cell Research

    doi: 10.1038/cr.2012.179

    IL-17 + MSCs showed reduced TGF-β1 expression. (A , B) When IL-17 was knocked down by siRNA, the capacity to upregulate Tregs (A) and downregulate Th17 cells (B) in IL-17 + MSCs (#3), but not in IL-17 − MSCs (#5), was increased.
    Figure Legend Snippet: IL-17 + MSCs showed reduced TGF-β1 expression. (A , B) When IL-17 was knocked down by siRNA, the capacity to upregulate Tregs (A) and downregulate Th17 cells (B) in IL-17 + MSCs (#3), but not in IL-17 − MSCs (#5), was increased.

    Techniques Used: Expressing

    IL-17 downregulated TGF-β1 expression via activation of NF-κB pathway. (A , B) Recombinant IL-17 (20 ng/ml) treatment attenuated bulk and IL-17– MSC (#5)-mediated upregulation of Tregs (A) and downregulation of Th17 cells
    Figure Legend Snippet: IL-17 downregulated TGF-β1 expression via activation of NF-κB pathway. (A , B) Recombinant IL-17 (20 ng/ml) treatment attenuated bulk and IL-17– MSC (#5)-mediated upregulation of Tregs (A) and downregulation of Th17 cells

    Techniques Used: Expressing, Activation Assay, Recombinant

    20) Product Images from "Soy isoflavones reduce asthma exacerbation in asthmatics with high PAI-1 producing genotypes"

    Article Title: Soy isoflavones reduce asthma exacerbation in asthmatics with high PAI-1 producing genotypes

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2019.01.020

    TGF-β1-induced production of PAI-1 gene and protein in normal human bronchial epithelial (NHBE) cells. NHBE cells were cultured and stimulated with recombinant human TGF-β1 (2 ng/ml) with and without genistein pretreatment (1 μM or 5 μM) for 1 hour. Cell pellets were used for real time PCR and cell culture supernatants for PAI-1 protein ELISA.
    Figure Legend Snippet: TGF-β1-induced production of PAI-1 gene and protein in normal human bronchial epithelial (NHBE) cells. NHBE cells were cultured and stimulated with recombinant human TGF-β1 (2 ng/ml) with and without genistein pretreatment (1 μM or 5 μM) for 1 hour. Cell pellets were used for real time PCR and cell culture supernatants for PAI-1 protein ELISA.

    Techniques Used: Cell Culture, Recombinant, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    21) Product Images from "Treatment of renal fibrosis by rebalancing TGF-β/Smad signaling with the combination of asiatic acid and naringenin"

    Article Title: Treatment of renal fibrosis by rebalancing TGF-β/Smad signaling with the combination of asiatic acid and naringenin

    Journal: Oncotarget

    doi:

    Combination treatment with AA and NG produces a further suppressive effect on renal TGF-β1 and miR-21 expression in the UUO kidney a. Immunohistochemistry and real-time PCR show the inhibitory effect of AA, NG, and their combination (CB) on TGF-β1 expression in the UUO kidney. b. , c. Real-time PCR detects that the combination treatment with AA and NG results in a further suppression of miR-21 expression in the UUO kidney and in cultured TECs. d. , e. Real-time PCR detects miR-29b expression in the UUO kidney and in cultured TECs. Data represent the mean ± SEM for groups of 6-8 mice. * p
    Figure Legend Snippet: Combination treatment with AA and NG produces a further suppressive effect on renal TGF-β1 and miR-21 expression in the UUO kidney a. Immunohistochemistry and real-time PCR show the inhibitory effect of AA, NG, and their combination (CB) on TGF-β1 expression in the UUO kidney. b. , c. Real-time PCR detects that the combination treatment with AA and NG results in a further suppression of miR-21 expression in the UUO kidney and in cultured TECs. d. , e. Real-time PCR detects miR-29b expression in the UUO kidney and in cultured TECs. Data represent the mean ± SEM for groups of 6-8 mice. * p

    Techniques Used: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Cell Culture, Mouse Assay

    Combination of AA and NG produces an additive effect on inhibition of TGF-β/Smad signaling via differential mechanisms in vitro a. Effect of AA, NG, and their combination (CB) on TGF-β1(2ng/ml)-induced mRNA expression of Smad3, Smad7, and TGF-β1 by real-time PCR. b. Effect of AA, NG, and their combination (CB) on TGF-β1(2ng/ml)-induced protein levels of phospho-Smad3, Smad3, Smad7, and Smurf2 by Western blot analysis. Note that while AA inhibits phosphorylation of Smad3 by induction of Smad7, NG blocks Smad3 signaling by inhibiting Smad3 mRNA and protein expression. The combination of AA and NG additively inhibits TGF-β/Smad3 signaling. Data represent the mean ± SEM for at least 3 independent experiments. * p
    Figure Legend Snippet: Combination of AA and NG produces an additive effect on inhibition of TGF-β/Smad signaling via differential mechanisms in vitro a. Effect of AA, NG, and their combination (CB) on TGF-β1(2ng/ml)-induced mRNA expression of Smad3, Smad7, and TGF-β1 by real-time PCR. b. Effect of AA, NG, and their combination (CB) on TGF-β1(2ng/ml)-induced protein levels of phospho-Smad3, Smad3, Smad7, and Smurf2 by Western blot analysis. Note that while AA inhibits phosphorylation of Smad3 by induction of Smad7, NG blocks Smad3 signaling by inhibiting Smad3 mRNA and protein expression. The combination of AA and NG additively inhibits TGF-β/Smad3 signaling. Data represent the mean ± SEM for at least 3 independent experiments. * p

    Techniques Used: Inhibition, In Vitro, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Combination of AA and NG produces a better protective effect on TGF-β1-induced fibrotic response in vitro a. Effect of AA, NG, or their combination on TGF-β1 (2ng/ml)-induced mRNA expression of collagen I and α-SMA detected by real-time PCR. b. Effect of AA, NG, or their combination on TGF-β1 (2ng/ml)-induced mRNA expression of collagen I and α-SMA detected by Western blot analysis. Data represent the mean ± SEM for at least 3 independent experiments. ** p
    Figure Legend Snippet: Combination of AA and NG produces a better protective effect on TGF-β1-induced fibrotic response in vitro a. Effect of AA, NG, or their combination on TGF-β1 (2ng/ml)-induced mRNA expression of collagen I and α-SMA detected by real-time PCR. b. Effect of AA, NG, or their combination on TGF-β1 (2ng/ml)-induced mRNA expression of collagen I and α-SMA detected by Western blot analysis. Data represent the mean ± SEM for at least 3 independent experiments. ** p

    Techniques Used: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    22) Product Images from "Renal Fibrosis "

    Article Title: Renal Fibrosis

    Journal: The American Journal of Pathology

    doi:

    TGF-β1 mRNA expression is up-regulated in EMT. Induction of EMT by TGF-β 1 /EGF or with type IV collagen α1NC1 domain results in up-regulation of TGF-β 1 mRNA expression ( A ). Induction with TGF-β 1 /EGF resulted in a peak after 6 hours, whereas after incubation with type IV collagen α1NC1 domains resulted in an up-regulation after 24 hours ( B ) summarizes the densitometric analysis of experiments with TGF-β 1 /EGF ( left ) and with α1NC1 domain ( right ). Treatment of MCT cells with TGF-β 1 and EGF results in an increase in TGF-β 1 mRNA after 6 hours by (291 ± 48% compared to K1 control), and remains elevated after 12 hours (204 ± 34%) and 24 hours (162 ± 18%). Treatment with α1NC1 domain leads to a significant increase in TGF-β 1 mRNA after 24 hours (175.2 ± 24%), however results after 6 hours (110.7 ± 8%) and 12 hours (125.7 ± 9%) were not significant. In solid-phase direct ELISA for FSP-1 ( C ) co-incubation of α1NC1 domain with neutralizing antibodies to TGF-β 1 (MCT NC1 anti-TGF) reduced the increase in FSP-1 expression significantly (158.1 ± 23% compared to K1 control), whereas the addition of neutralizing EGF antibodies (MCT NC1 anti-EGF) had no significant effect (193.5 ± 29%). Addition of neutralizing antibodies to TGF and EGF (MCT TE anti-TE) abolished growth factor-induced EMT (111.8 ± 14%). These results suggest a role for TGF-β 1 autocrine stimulation for mediation of EMT. *, P
    Figure Legend Snippet: TGF-β1 mRNA expression is up-regulated in EMT. Induction of EMT by TGF-β 1 /EGF or with type IV collagen α1NC1 domain results in up-regulation of TGF-β 1 mRNA expression ( A ). Induction with TGF-β 1 /EGF resulted in a peak after 6 hours, whereas after incubation with type IV collagen α1NC1 domains resulted in an up-regulation after 24 hours ( B ) summarizes the densitometric analysis of experiments with TGF-β 1 /EGF ( left ) and with α1NC1 domain ( right ). Treatment of MCT cells with TGF-β 1 and EGF results in an increase in TGF-β 1 mRNA after 6 hours by (291 ± 48% compared to K1 control), and remains elevated after 12 hours (204 ± 34%) and 24 hours (162 ± 18%). Treatment with α1NC1 domain leads to a significant increase in TGF-β 1 mRNA after 24 hours (175.2 ± 24%), however results after 6 hours (110.7 ± 8%) and 12 hours (125.7 ± 9%) were not significant. In solid-phase direct ELISA for FSP-1 ( C ) co-incubation of α1NC1 domain with neutralizing antibodies to TGF-β 1 (MCT NC1 anti-TGF) reduced the increase in FSP-1 expression significantly (158.1 ± 23% compared to K1 control), whereas the addition of neutralizing EGF antibodies (MCT NC1 anti-EGF) had no significant effect (193.5 ± 29%). Addition of neutralizing antibodies to TGF and EGF (MCT TE anti-TE) abolished growth factor-induced EMT (111.8 ± 14%). These results suggest a role for TGF-β 1 autocrine stimulation for mediation of EMT. *, P

    Techniques Used: Expressing, Incubation, Direct ELISA

    23) Product Images from "TGFBR2 mutations alter smooth muscle cell phenotype and predispose to thoracic aortic aneurysms and dissections"

    Article Title: TGFBR2 mutations alter smooth muscle cell phenotype and predispose to thoracic aortic aneurysms and dissections

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvq230

    Defective transformation of quiescent fibroblasts to myofibroblasts with TGF-β in patient and control fibroblasts. ( A ) Q-PCR analysis of mRNA expression of SMC contractile protein genes in fibroblasts various times after exposure to TGF-β1
    Figure Legend Snippet: Defective transformation of quiescent fibroblasts to myofibroblasts with TGF-β in patient and control fibroblasts. ( A ) Q-PCR analysis of mRNA expression of SMC contractile protein genes in fibroblasts various times after exposure to TGF-β1

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, Expressing

    24) Product Images from "Excessive activation of the TLR9/TGF-β1/PDGF-B pathway in the peripheral blood of patients with systemic lupus erythematosus"

    Article Title: Excessive activation of the TLR9/TGF-β1/PDGF-B pathway in the peripheral blood of patients with systemic lupus erythematosus

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-017-1238-8

    Transforming growth factor-β1 ( TGF- β 1 ) induces the production of platelet-derived growth factor-B ( PDGF-B ) in vitro. Protein levels of PDGF-B in blood cell cultures of SLE patients with or without 2.5 ng/ml recombinant protein TGF-β1 ( a ; N SLE = 7), and with or without TGF-β1 antagonists (5 μM SB431542, 1 μg/ml αTGF-β1) ( b ; N SLE = 6). The mRNA expression of PDGF-B in blood cell cultures of SLE patients with or without 2.5 ng/ml recombinant protein TGF-β1 ( c ; N SLE = 10), and with or without TGF-β1 antagonists (5 μM SB431542, 1 μg/ml αTGF-β1) ( d ; N SLE = 9). The mRNA expression of PDGF-B in blood cultures of SLE patients with 500 nM CpG, 5 μM SB431542, or 500 nM CpG plus 5 μM SB431542 ( e ; N SLE = 16). Cells were cultured for 24 h, and then mRNA expressions were detected by qPCR. The results are presented as mean and SEM. GAPDH glyceraldehyde-3-phosphate dehydrogenase
    Figure Legend Snippet: Transforming growth factor-β1 ( TGF- β 1 ) induces the production of platelet-derived growth factor-B ( PDGF-B ) in vitro. Protein levels of PDGF-B in blood cell cultures of SLE patients with or without 2.5 ng/ml recombinant protein TGF-β1 ( a ; N SLE = 7), and with or without TGF-β1 antagonists (5 μM SB431542, 1 μg/ml αTGF-β1) ( b ; N SLE = 6). The mRNA expression of PDGF-B in blood cell cultures of SLE patients with or without 2.5 ng/ml recombinant protein TGF-β1 ( c ; N SLE = 10), and with or without TGF-β1 antagonists (5 μM SB431542, 1 μg/ml αTGF-β1) ( d ; N SLE = 9). The mRNA expression of PDGF-B in blood cultures of SLE patients with 500 nM CpG, 5 μM SB431542, or 500 nM CpG plus 5 μM SB431542 ( e ; N SLE = 16). Cells were cultured for 24 h, and then mRNA expressions were detected by qPCR. The results are presented as mean and SEM. GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Techniques Used: Derivative Assay, In Vitro, Recombinant, Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Levels of Toll-like receptor 9 ( TLR9 ), transforming growth factor-β1 ( TGF- β 1 ), and platelet-derived growth factor-B ( PDGF-B ) and proteinuria in SLE patients with LN. Correlations between mRNA expression of TGF-β1 and PDGF-B ( a ; N LN = 38). Correlations between mRNA expression of TLR9 and TGF-β1 ( b ; N LN = 38). Correlations between mRNA expression of TLR9 and PDGF-B ( c ; N LN = 38). Correlations between protein levels of TGF-β1 and PDGF-B ( d ; N LN = 38). Correlations between protein levels of TGF-β1 and PDGF-B ( d ; N LN = 38). Correlations between protein levels of PDGF-B and urine protein ( e ; N LN = 38). GAPDH glyceraldehyde-3-phosphate dehydrogenase
    Figure Legend Snippet: Levels of Toll-like receptor 9 ( TLR9 ), transforming growth factor-β1 ( TGF- β 1 ), and platelet-derived growth factor-B ( PDGF-B ) and proteinuria in SLE patients with LN. Correlations between mRNA expression of TGF-β1 and PDGF-B ( a ; N LN = 38). Correlations between mRNA expression of TLR9 and TGF-β1 ( b ; N LN = 38). Correlations between mRNA expression of TLR9 and PDGF-B ( c ; N LN = 38). Correlations between protein levels of TGF-β1 and PDGF-B ( d ; N LN = 38). Correlations between protein levels of TGF-β1 and PDGF-B ( d ; N LN = 38). Correlations between protein levels of PDGF-B and urine protein ( e ; N LN = 38). GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Techniques Used: Derivative Assay, Expressing

    Levels of Toll-like receptor 9 ( TLR9 ), transforming growth factor-β1 ( TGF- β 1 ), and platelet-derived growth factor-B ( PDGF-B ) in the peripheral blood of systemic lupus erythematosus ( SLE ) patients. The mRNA expression of TLR9 ( a ) in the peripheral blood of SLE patients ( N SLE = 112) and healthy controls ( N Control = 49). Levels of TGF-β1 ( b ) and PDGF-B ( c ) in the plasma of SLE patients ( N SLE = 77) and healthy controls ( N Control = 56). Levels of SLE Disease Activity Index ( SLEDAI ) ( d ), TGF-β1 ( e ), and PDGF-B ( f ) in the same group of SLE patients ( N SLE = 20) before and after immunosuppressive treatment. The results in a.b.c. are presented as Mean and SEM. GAPDH glyceraldehyde-3-phosphate dehydrogenase
    Figure Legend Snippet: Levels of Toll-like receptor 9 ( TLR9 ), transforming growth factor-β1 ( TGF- β 1 ), and platelet-derived growth factor-B ( PDGF-B ) in the peripheral blood of systemic lupus erythematosus ( SLE ) patients. The mRNA expression of TLR9 ( a ) in the peripheral blood of SLE patients ( N SLE = 112) and healthy controls ( N Control = 49). Levels of TGF-β1 ( b ) and PDGF-B ( c ) in the plasma of SLE patients ( N SLE = 77) and healthy controls ( N Control = 56). Levels of SLE Disease Activity Index ( SLEDAI ) ( d ), TGF-β1 ( e ), and PDGF-B ( f ) in the same group of SLE patients ( N SLE = 20) before and after immunosuppressive treatment. The results in a.b.c. are presented as Mean and SEM. GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Techniques Used: Derivative Assay, Expressing, Activity Assay

    Correlations among levels of Toll-like receptor 9 ( TLR9 ), transforming growth factor-β1 ( TGF- β 1 ), and platelet-derived growth factor-B ( PDGF-B ) in human peripheral blood. Correlations between protein levels of TGF-β1 and PDGF-B in healthy controls ( a ; N Control = 56) and systemic lupus erythematosus ( SLE ) patients ( b ; N SLE = 79). Correlations between mRNA expression of TGF-β1 and PDGF-B in healthy controls ( c ; N Control = 46) and SLE patients ( d ; N SLE = 106). Correlations between TLR9 and TGF-β1 ( e , f ), or between TLR9 and PDGF-B ( g , h ). The mRNA expression of healthy controls ( e , g ; N Control = 46) and that of SLE patients ( f , h ; N SLE = 106). GAPDH glyceraldehyde-3-phosphate dehydrogenase
    Figure Legend Snippet: Correlations among levels of Toll-like receptor 9 ( TLR9 ), transforming growth factor-β1 ( TGF- β 1 ), and platelet-derived growth factor-B ( PDGF-B ) in human peripheral blood. Correlations between protein levels of TGF-β1 and PDGF-B in healthy controls ( a ; N Control = 56) and systemic lupus erythematosus ( SLE ) patients ( b ; N SLE = 79). Correlations between mRNA expression of TGF-β1 and PDGF-B in healthy controls ( c ; N Control = 46) and SLE patients ( d ; N SLE = 106). Correlations between TLR9 and TGF-β1 ( e , f ), or between TLR9 and PDGF-B ( g , h ). The mRNA expression of healthy controls ( e , g ; N Control = 46) and that of SLE patients ( f , h ; N SLE = 106). GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Techniques Used: Derivative Assay, Expressing

    CpG induces upregulation of transforming growth factor-β1 ( TGF- β 1 ) and platelet-derived growth factor-B ( PDGF-B ) in vitro. Blood cells from healthy controls ( a , b ; N Control = 12) and systemic lupus erythematosus ( SLE ) patients ( c , d ; N SLE = 16) were stimulated with or without 500 nM CpG for 24 h, and then the mRNA expression of TGF-β1 and PDGF-B were detected by qPCR. Multiple rates of mRNA expression of TGF-β1 ( e ) and PDGF-B ( f ) in healthy controls ( N Control = 12) and SLE patients ( N SLE = 16) were calculated as CpG/Media. The results were presented as mean and SEM. GAPDH glyceraldehyde-3-phosphate dehydrogenase
    Figure Legend Snippet: CpG induces upregulation of transforming growth factor-β1 ( TGF- β 1 ) and platelet-derived growth factor-B ( PDGF-B ) in vitro. Blood cells from healthy controls ( a , b ; N Control = 12) and systemic lupus erythematosus ( SLE ) patients ( c , d ; N SLE = 16) were stimulated with or without 500 nM CpG for 24 h, and then the mRNA expression of TGF-β1 and PDGF-B were detected by qPCR. Multiple rates of mRNA expression of TGF-β1 ( e ) and PDGF-B ( f ) in healthy controls ( N Control = 12) and SLE patients ( N SLE = 16) were calculated as CpG/Media. The results were presented as mean and SEM. GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Techniques Used: Derivative Assay, In Vitro, Expressing, Real-time Polymerase Chain Reaction

    25) Product Images from "Corneal myofibroblasts inhibit regenerating nerves during wound healing"

    Article Title: Corneal myofibroblasts inhibit regenerating nerves during wound healing

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30964-y

    Critical role of TGF-β1 and its receptor in mediating the inhibitory effect of myofibroblasts (Myos) on neurite outgrowth in 1 day old cultures. ( A ) Phase contrast photograph of plated ND7/23 cells (highly refractile, round cell bodies) and co-cultured Myos (examples arrowed) in the presence of DMSO or the TGF-β receptor inhibitor SB431542. Note the rare neurites expressed by ND7/23 cells after 1 day in co-culture with Myos and DMSO, in contrast with the numerous neurites expressed by ND cells, in the same co-culture, when SB431542 is present. ( B ) Plot of the percentage of plated ND7/23 cells with neurites > 40 µm long in ND + Myo co-cultures in the presence or absence of SB431542. See Fig. S3B for sample Western blot made from co-cultures in this experiment, confirming the presence of similar amounts of Tuj-1 and of α-SMA positive myofibroblasts irrespective of treatment with SB431542. ( C ) Phase contrast photograph of ND7/23 cells (highly refractile, round cell bodies) in pure cultures in the presence of DMSO or TGF-β1. Note the multiple neurites expressed by ND7/23 cells after 1 day in culture with DMSO, and the marked decrease in neurites when ND cells are treated with TGF-β1. ( D ) Plot of the percentage of plated ND7/23 cells with neurites > 40 µm long when treated with or without TGF-β1. The dependence of this effect on activation of the TGF-β receptor is demonstrated by the ability of the TGF-β receptor inhibitor SB431542 to eliminate the anti-neuritogenic impact of TGF-β1. See Fig. S3C for sample Western blot made from mono-cultures in this experiment, confirming the presence of similar amounts of Tuj-1 irrespective of treatment with DMSO, TGF-β1, SB431542 or TGF-β1 + SB431542. All values in graphs are means ± SD from 3 separate experiments. * P
    Figure Legend Snippet: Critical role of TGF-β1 and its receptor in mediating the inhibitory effect of myofibroblasts (Myos) on neurite outgrowth in 1 day old cultures. ( A ) Phase contrast photograph of plated ND7/23 cells (highly refractile, round cell bodies) and co-cultured Myos (examples arrowed) in the presence of DMSO or the TGF-β receptor inhibitor SB431542. Note the rare neurites expressed by ND7/23 cells after 1 day in co-culture with Myos and DMSO, in contrast with the numerous neurites expressed by ND cells, in the same co-culture, when SB431542 is present. ( B ) Plot of the percentage of plated ND7/23 cells with neurites > 40 µm long in ND + Myo co-cultures in the presence or absence of SB431542. See Fig. S3B for sample Western blot made from co-cultures in this experiment, confirming the presence of similar amounts of Tuj-1 and of α-SMA positive myofibroblasts irrespective of treatment with SB431542. ( C ) Phase contrast photograph of ND7/23 cells (highly refractile, round cell bodies) in pure cultures in the presence of DMSO or TGF-β1. Note the multiple neurites expressed by ND7/23 cells after 1 day in culture with DMSO, and the marked decrease in neurites when ND cells are treated with TGF-β1. ( D ) Plot of the percentage of plated ND7/23 cells with neurites > 40 µm long when treated with or without TGF-β1. The dependence of this effect on activation of the TGF-β receptor is demonstrated by the ability of the TGF-β receptor inhibitor SB431542 to eliminate the anti-neuritogenic impact of TGF-β1. See Fig. S3C for sample Western blot made from mono-cultures in this experiment, confirming the presence of similar amounts of Tuj-1 irrespective of treatment with DMSO, TGF-β1, SB431542 or TGF-β1 + SB431542. All values in graphs are means ± SD from 3 separate experiments. * P

    Techniques Used: Cell Culture, Co-Culture Assay, Western Blot, Activation Assay

    TGF-β1 increases relative p-CRMP2 expression in pure ND7/23 cultures. ( A ) Representative Western blots showing protein levels for phosphorylated (p-) CRMP2 and total (t-) CRMP2 in cultured ND7/23 cells with or without TGF-β1 stimulation. The samples were run on separate blots. Basal levels of both p- and t-CRMP2 were distinctly above zero. After 1 hour, TGF-β1 increased the expression of p-CRMP2, but not that of t-CRMP2. The TGF-β receptor inhibitor SB431542 blocked the up-regulation of p-CRMP2 observed following TGF-β1 stimulation. ( B ) Plot of relative expression of p-CRMP2/t-CRMP2 normalized to densitometric values obtained in cells treated with TGF-β1. Data shown are averaged over three experiments. Total CRMP2 levels were were used as loading controls. Data are expressed as means ± SD. ** P
    Figure Legend Snippet: TGF-β1 increases relative p-CRMP2 expression in pure ND7/23 cultures. ( A ) Representative Western blots showing protein levels for phosphorylated (p-) CRMP2 and total (t-) CRMP2 in cultured ND7/23 cells with or without TGF-β1 stimulation. The samples were run on separate blots. Basal levels of both p- and t-CRMP2 were distinctly above zero. After 1 hour, TGF-β1 increased the expression of p-CRMP2, but not that of t-CRMP2. The TGF-β receptor inhibitor SB431542 blocked the up-regulation of p-CRMP2 observed following TGF-β1 stimulation. ( B ) Plot of relative expression of p-CRMP2/t-CRMP2 normalized to densitometric values obtained in cells treated with TGF-β1. Data shown are averaged over three experiments. Total CRMP2 levels were were used as loading controls. Data are expressed as means ± SD. ** P

    Techniques Used: Expressing, Western Blot, Cell Culture

    26) Product Images from "Dual roles of parathyroid hormone related protein in TGF-β1 signaling and fibronectin up-regulation in mesangial cells"

    Article Title: Dual roles of parathyroid hormone related protein in TGF-β1 signaling and fibronectin up-regulation in mesangial cells

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171061

    Knocking down PTH1R expression with siRNA reversed the blocking effect of PTHrP on TGF-β1 and HG-mediated Smad2/3 activation and FN up-regulation at 3 h, but not at 24 h Subconfluent MCs were transfected with 100 nM rat PTH1R siRNA or control siRNA. Cells were then treated with TGF-β1 or HG for the indicated duration, with or without PTHrP. The protein levels of p-Smad2/3 and FN were assessed by Western blot. ( A )* P
    Figure Legend Snippet: Knocking down PTH1R expression with siRNA reversed the blocking effect of PTHrP on TGF-β1 and HG-mediated Smad2/3 activation and FN up-regulation at 3 h, but not at 24 h Subconfluent MCs were transfected with 100 nM rat PTH1R siRNA or control siRNA. Cells were then treated with TGF-β1 or HG for the indicated duration, with or without PTHrP. The protein levels of p-Smad2/3 and FN were assessed by Western blot. ( A )* P

    Techniques Used: Expressing, Blocking Assay, Activation Assay, Transfection, Western Blot

    Short-term stimulation of PTHrP prevented TGF-β1/Smad signaling and FN up-regulation, whereas long-term treatment of PTHrP did not prevent TGF-β1-induced Smad2/3 phosphorylation and FN up-regulation MCs were pretreated with PTHrP (100 nM, 30 min) before TGF-β1 treatment for the indicated periods. ( A , C ) Smad2/3 phosphorylation was detected by Western blot (* P
    Figure Legend Snippet: Short-term stimulation of PTHrP prevented TGF-β1/Smad signaling and FN up-regulation, whereas long-term treatment of PTHrP did not prevent TGF-β1-induced Smad2/3 phosphorylation and FN up-regulation MCs were pretreated with PTHrP (100 nM, 30 min) before TGF-β1 treatment for the indicated periods. ( A , C ) Smad2/3 phosphorylation was detected by Western blot (* P

    Techniques Used: Western Blot

    27) Product Images from "miR-185 Inhibits Fibrogenic Activation of Hepatic Stellate Cells and Prevents Liver Fibrosis"

    Article Title: miR-185 Inhibits Fibrogenic Activation of Hepatic Stellate Cells and Prevents Liver Fibrosis

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.11.010

    Knockdown of Endogenous miR-185 Promoted Basal/TGF-β1-Induced Liver Fibrosis LX-2 cells were transfected with inhibitor-NC or miR-185 inhibitor for 48 hr; inhibition of miR-185 in LX-2 cells induced liver fibrogenesis-related gene transcription (A) and translocation (B). (C) Western blot data were quantified. (D) TGF-β1-induced liver fibrosis, and miR-185 inhibitors (anti-miR-185) upregulated protein expression levels of COL I, COL III, and α-SMA after TGF-β1 further induced HSC activation. Three independent experiments gave similar results (mean ± SEM, n = 3, *p
    Figure Legend Snippet: Knockdown of Endogenous miR-185 Promoted Basal/TGF-β1-Induced Liver Fibrosis LX-2 cells were transfected with inhibitor-NC or miR-185 inhibitor for 48 hr; inhibition of miR-185 in LX-2 cells induced liver fibrogenesis-related gene transcription (A) and translocation (B). (C) Western blot data were quantified. (D) TGF-β1-induced liver fibrosis, and miR-185 inhibitors (anti-miR-185) upregulated protein expression levels of COL I, COL III, and α-SMA after TGF-β1 further induced HSC activation. Three independent experiments gave similar results (mean ± SEM, n = 3, *p

    Techniques Used: Transfection, Inhibition, Translocation Assay, Western Blot, Expressing, Activation Assay

    miR-185 Is Abundant in LX-2 Cells but Downregulated in Activated LX-2 Cells The human hepatic stellate cell (HSC) cell line LX-2 was treated with 5.0 ng/mL transforming growth factor β-1 (TGF-β1) for 0, 3, 6, 12, or 24 hr or with 2.5, 5.0, or 7.5 ng/mL TGF-β1 for 24.0 hr. (A) mRNA expression of key genes involved in the activation of HSC α-SMA, COL1A1, COL1A2, and COL3A1 was analyzed by real-time PCR. All genes increased in a time-dependent manner. mRNA levels of Ras homolog enriched in brain (RHEB) and rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR) were assessed by real-time PCR. When HSCs were activated by TGF-β1 with a different dose (B) or time (C), RHEB and RICTOR were upregulated in mRNA levels. (D) Protein expressions of α-SMA, COL I, COL III, RHEB, and RICTOR was determined by western blotting; GAPDH or β-actin was used as a loading control. When HSCs were activated, all genes were upregulated in protein levels. miR-185-5p expression was reduced in activated HSCs in a dose-dependent manner (E) or time-dependent manner (F) examined by real-time PCR (n = 3, *p
    Figure Legend Snippet: miR-185 Is Abundant in LX-2 Cells but Downregulated in Activated LX-2 Cells The human hepatic stellate cell (HSC) cell line LX-2 was treated with 5.0 ng/mL transforming growth factor β-1 (TGF-β1) for 0, 3, 6, 12, or 24 hr or with 2.5, 5.0, or 7.5 ng/mL TGF-β1 for 24.0 hr. (A) mRNA expression of key genes involved in the activation of HSC α-SMA, COL1A1, COL1A2, and COL3A1 was analyzed by real-time PCR. All genes increased in a time-dependent manner. mRNA levels of Ras homolog enriched in brain (RHEB) and rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR) were assessed by real-time PCR. When HSCs were activated by TGF-β1 with a different dose (B) or time (C), RHEB and RICTOR were upregulated in mRNA levels. (D) Protein expressions of α-SMA, COL I, COL III, RHEB, and RICTOR was determined by western blotting; GAPDH or β-actin was used as a loading control. When HSCs were activated, all genes were upregulated in protein levels. miR-185-5p expression was reduced in activated HSCs in a dose-dependent manner (E) or time-dependent manner (F) examined by real-time PCR (n = 3, *p

    Techniques Used: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot

    Schematic TGF-β1-induced activated HSCs also downregulate miR-185 concomitant with increased expression of RHEB and RICTOR. miR-185 prevents liver fibrogenesis by inhibiting HSC activation through inhibiting RHEB and RICTOR.
    Figure Legend Snippet: Schematic TGF-β1-induced activated HSCs also downregulate miR-185 concomitant with increased expression of RHEB and RICTOR. miR-185 prevents liver fibrogenesis by inhibiting HSC activation through inhibiting RHEB and RICTOR.

    Techniques Used: Expressing, Activation Assay

    miR-185-Suppressed HSC Activation and Basal/TGF-β1-Induced Fibrous ECM Production of LX-2 Cells LX-2 cells were seeded into six-well plates at a density of 1.0 × 10 6 cells per well, and cells were transiently transfected overnight with miR-185 mimics (miR-185) or negative mimic controls (miR-NC). (A and B) After 48 hr, (A) real-time qPCR was performed to analyze the mRNA levels, and (B) western blotting was performed to analyze the protein levels of liver fibrogenesis-related genes. miR-185 mimics downregulated mRNA and protein expression levels of COL I, COL III, and α-SMA. (C) Western blotting data were quantified using the ImageJ software. (D) To detect the effects of miR-185 on TGF-β1-induced profibrogenic gene expression, LX-2 cells were transfected with miR-185 mimics for 24 hr and then replaced with new culture medium treated with or without 5.0 ng/mL TGF-β1 for another 24 hr. α-SMA, COL I, and COL III protein levels were detected by western blotting. miR-185 can repress phenotypes associated with TGF-β1-induced transition of resident fibroblasts. The results are representative of three independent experiments and are presented as mean ± SEM (n = 3, *p
    Figure Legend Snippet: miR-185-Suppressed HSC Activation and Basal/TGF-β1-Induced Fibrous ECM Production of LX-2 Cells LX-2 cells were seeded into six-well plates at a density of 1.0 × 10 6 cells per well, and cells were transiently transfected overnight with miR-185 mimics (miR-185) or negative mimic controls (miR-NC). (A and B) After 48 hr, (A) real-time qPCR was performed to analyze the mRNA levels, and (B) western blotting was performed to analyze the protein levels of liver fibrogenesis-related genes. miR-185 mimics downregulated mRNA and protein expression levels of COL I, COL III, and α-SMA. (C) Western blotting data were quantified using the ImageJ software. (D) To detect the effects of miR-185 on TGF-β1-induced profibrogenic gene expression, LX-2 cells were transfected with miR-185 mimics for 24 hr and then replaced with new culture medium treated with or without 5.0 ng/mL TGF-β1 for another 24 hr. α-SMA, COL I, and COL III protein levels were detected by western blotting. miR-185 can repress phenotypes associated with TGF-β1-induced transition of resident fibroblasts. The results are representative of three independent experiments and are presented as mean ± SEM (n = 3, *p

    Techniques Used: Activation Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Software

    28) Product Images from "LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells"

    Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21953

    Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.
    Figure Legend Snippet: Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.

    Techniques Used: Isolation, Quantitative RT-PCR, Transfection, Expressing

    Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.
    Figure Legend Snippet: Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.

    Techniques Used: Transformation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection

    Impact of LINE-1 ORF-1 siRNAs on EMT Programming Cells were transfected with two unique target-specific siRNAs to LINE-1 targeting ORF1 regions or control siRNA (scramble) or no siRNA (mock), Forty-eight hours post-transfection, cells were challenged with 3 ng/ml TGF-β1 for an additional 48 hours or control. Whole cell lysates were analyzed by immunoblotting for ORF1p, E-Cadherin, vimentin or GAPDH.
    Figure Legend Snippet: Impact of LINE-1 ORF-1 siRNAs on EMT Programming Cells were transfected with two unique target-specific siRNAs to LINE-1 targeting ORF1 regions or control siRNA (scramble) or no siRNA (mock), Forty-eight hours post-transfection, cells were challenged with 3 ng/ml TGF-β1 for an additional 48 hours or control. Whole cell lysates were analyzed by immunoblotting for ORF1p, E-Cadherin, vimentin or GAPDH.

    Techniques Used: Transfection

    LINE-1 Modulates the Anti-proliferative Activity of TGF-β1 and Receptor Tyrosine Kinase Inhibitors Clones constitutively expressing wild type L1 (L1), a mutant L1 (mut L1), or empty vector were treated with various concentrations of TGF-β1 or control ( A ), Receptor tyrosine kinase inhibitors: sunitinib malate (VEGFR2, PDGFRβ and c-KIT inhibitor) ( B ), EGFR inhibitors- Erlotinib ( C ) and Gefitinib ( D ). After 72 h of treatment, proliferation was determined by the MTT assay and normalized to untreated or DMSO (vehicle for receptor tyrosine kinase inhibitors) for each cell type. Data represent the mean plus SEM for individual samples from three independent experiments. ( E ) Stably transfected cells were serum-starved for 24 h and cell lysates analyzed by immunoblotting for phospho-ERK1/2 (p-Erk1/2), phospho-AKT1 (p-Akt), ERK1/2 or AKT1 (Akt). Data are representative two independent experiments using clones #5 (wild type L1) and #13 (mutant L1).
    Figure Legend Snippet: LINE-1 Modulates the Anti-proliferative Activity of TGF-β1 and Receptor Tyrosine Kinase Inhibitors Clones constitutively expressing wild type L1 (L1), a mutant L1 (mut L1), or empty vector were treated with various concentrations of TGF-β1 or control ( A ), Receptor tyrosine kinase inhibitors: sunitinib malate (VEGFR2, PDGFRβ and c-KIT inhibitor) ( B ), EGFR inhibitors- Erlotinib ( C ) and Gefitinib ( D ). After 72 h of treatment, proliferation was determined by the MTT assay and normalized to untreated or DMSO (vehicle for receptor tyrosine kinase inhibitors) for each cell type. Data represent the mean plus SEM for individual samples from three independent experiments. ( E ) Stably transfected cells were serum-starved for 24 h and cell lysates analyzed by immunoblotting for phospho-ERK1/2 (p-Erk1/2), phospho-AKT1 (p-Akt), ERK1/2 or AKT1 (Akt). Data are representative two independent experiments using clones #5 (wild type L1) and #13 (mutant L1).

    Techniques Used: Activity Assay, Clone Assay, Expressing, Mutagenesis, Plasmid Preparation, MTT Assay, Stable Transfection, Transfection

    Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.
    Figure Legend Snippet: Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.

    Techniques Used: Activation Assay, Expressing, Isolation, Quantitative RT-PCR

    29) Product Images from "Astragaloside IV modulates TGF‐β1‐dependent epithelial‐mesenchymal transition in bleomycin‐induced pulmonary fibrosis. Astragaloside IV modulates TGF‐β1‐dependent epithelial‐mesenchymal transition in bleomycin‐induced pulmonary fibrosis"

    Article Title: Astragaloside IV modulates TGF‐β1‐dependent epithelial‐mesenchymal transition in bleomycin‐induced pulmonary fibrosis. Astragaloside IV modulates TGF‐β1‐dependent epithelial‐mesenchymal transition in bleomycin‐induced pulmonary fibrosis

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13725

    Astragaloside IV suppressed TGF ‐β1/ PI 3K/Akt induced FOXO 3a inactivity to reverse EMT in A549 cells. A, Western blots analysis of p‐ FOXO 3a (Thr32), p‐ FOXO 3a (Ser253), FOXO 3a, p‐Akt and Akt protein levels. β‐actin was used as the internal control. B, Relative expression of FOXO 3a mRNA level in A549. C, The expression levels of FOXO 3a protein were tested by western blot analysis. D, Immunostaining of FOXO 3a in A549 cells treated with 10 ng/mL TGF ‐β1, 100 μg/mL ASV , 10 μmol/L SB 431542 (a TGF ‐β1 inhibitor) and or 20 μmol/L LY 294002 (a PI 3K/Akt inhibitor). E, p‐ FOXO 3a (Thr32), p‐ FOXO 3a (Ser253), FOXO 3a, p‐Akt and Akt expression via western blot following treatment with PBS , ASV , TGF ‐β1, TGF ‐β1 + ASV , TGF ‐β1 + SB 431542 or TGF ‐β1 + LY 294002. F, Relative expression of FOXO 3a mRNA in groups transfected with pc DNA ‐3.1 + FOXO 3a or pc DNA ‐3.1 constructs. G, FOXO 3a protein expression following transfection with a FOXO 3a‐expressing plasmid. H, Western blots analysis of E‐cadherin and α‐ SMA in indicated groups. I, Double‐labelled immunofluorescent staining was performed to examine the expression of E‐cadherin (red) and α‐ SMA (green) in A549 cells in different groups. Scale bar = 50 μm * P
    Figure Legend Snippet: Astragaloside IV suppressed TGF ‐β1/ PI 3K/Akt induced FOXO 3a inactivity to reverse EMT in A549 cells. A, Western blots analysis of p‐ FOXO 3a (Thr32), p‐ FOXO 3a (Ser253), FOXO 3a, p‐Akt and Akt protein levels. β‐actin was used as the internal control. B, Relative expression of FOXO 3a mRNA level in A549. C, The expression levels of FOXO 3a protein were tested by western blot analysis. D, Immunostaining of FOXO 3a in A549 cells treated with 10 ng/mL TGF ‐β1, 100 μg/mL ASV , 10 μmol/L SB 431542 (a TGF ‐β1 inhibitor) and or 20 μmol/L LY 294002 (a PI 3K/Akt inhibitor). E, p‐ FOXO 3a (Thr32), p‐ FOXO 3a (Ser253), FOXO 3a, p‐Akt and Akt expression via western blot following treatment with PBS , ASV , TGF ‐β1, TGF ‐β1 + ASV , TGF ‐β1 + SB 431542 or TGF ‐β1 + LY 294002. F, Relative expression of FOXO 3a mRNA in groups transfected with pc DNA ‐3.1 + FOXO 3a or pc DNA ‐3.1 constructs. G, FOXO 3a protein expression following transfection with a FOXO 3a‐expressing plasmid. H, Western blots analysis of E‐cadherin and α‐ SMA in indicated groups. I, Double‐labelled immunofluorescent staining was performed to examine the expression of E‐cadherin (red) and α‐ SMA (green) in A549 cells in different groups. Scale bar = 50 μm * P

    Techniques Used: Western Blot, Expressing, Immunostaining, Transfection, Construct, Plasmid Preparation, Staining

    Astragaloside IV protected against bleomycin‐induced pulmonary fibrosis through suppressing TGF ‐β1‐mediated EMT . Astragaloside IV inhibited TGF ‐β1 induced activity of PI 3K/Akt pathway independent inactivity of FOXO 3a in pulmonary epithelial cell
    Figure Legend Snippet: Astragaloside IV protected against bleomycin‐induced pulmonary fibrosis through suppressing TGF ‐β1‐mediated EMT . Astragaloside IV inhibited TGF ‐β1 induced activity of PI 3K/Akt pathway independent inactivity of FOXO 3a in pulmonary epithelial cell

    Techniques Used: Activity Assay

    Astragaloside IV suppressed Bleomycin induced upregulation of TGF ‐β1 and hyperphosphorylation and inactivity of FoxO3a. A, relative expression of TGF ‐β1 mRNA in groups as measured by qPCR . B, Representative Western blots of TGF ‐β1 and densitometry quantified data of TGF ‐β1‐to‐β‐actin expression ratio. C, relative expression of FoxO3a mRNA in each group. D, Immunocytochemistry staining of FoxO3a in bleomycin or saline‐treated rats with or without ASV . E, Representative Western blots and densitometry quantified data of p‐ FOXO 3a (Thr32), p‐ FOXO 3a (Ser253) and FOXO 3a protein levels. β‐actin was used as the internal control. * P
    Figure Legend Snippet: Astragaloside IV suppressed Bleomycin induced upregulation of TGF ‐β1 and hyperphosphorylation and inactivity of FoxO3a. A, relative expression of TGF ‐β1 mRNA in groups as measured by qPCR . B, Representative Western blots of TGF ‐β1 and densitometry quantified data of TGF ‐β1‐to‐β‐actin expression ratio. C, relative expression of FoxO3a mRNA in each group. D, Immunocytochemistry staining of FoxO3a in bleomycin or saline‐treated rats with or without ASV . E, Representative Western blots and densitometry quantified data of p‐ FOXO 3a (Thr32), p‐ FOXO 3a (Ser253) and FOXO 3a protein levels. β‐actin was used as the internal control. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunocytochemistry, Staining

    Astragaloside IV inhibited TGF ‐β1 induced epithelial‐mesenchymal transition in pulmonary epithelial cells. A, TGF ‐β1 treatment induces more elongated morphological shape and increases scattering, while addition of ASV suppressed these changes. B, Immunofluorescence double staining showing the overlap of E‐cadherin (red) and α‐ SMA (green) in A549 cells in different groups. C, Western blots analysis of α‐ SMA and E‐cadherin protein in each group. * P
    Figure Legend Snippet: Astragaloside IV inhibited TGF ‐β1 induced epithelial‐mesenchymal transition in pulmonary epithelial cells. A, TGF ‐β1 treatment induces more elongated morphological shape and increases scattering, while addition of ASV suppressed these changes. B, Immunofluorescence double staining showing the overlap of E‐cadherin (red) and α‐ SMA (green) in A549 cells in different groups. C, Western blots analysis of α‐ SMA and E‐cadherin protein in each group. * P

    Techniques Used: Immunofluorescence, Double Staining, Western Blot

    30) Product Images from "Active Transforming Growth Factor-? in Wound Repair "

    Article Title: Active Transforming Growth Factor-? in Wound Repair

    Journal: The American Journal of Pathology

    doi:

    Time course of active TGF-β generation in incisional wound repair. Wound tissue, surrounding tissue, and normal skin were embedded in methyl cellulose. Four cryosections (20 μm) representing four wounds of one animal were prepared on duplicate coverslips and incubated overnight with MLECs. Twenty animals were used for this time course (two for each time point). MLECs were lysed and luciferase activity was determined and converted to TGF-β concentration (pg/mm 3 ) using a recombinant human TGF-β1 standard curve. The data represent mean values ± SEM of eight determinations in duplicate.
    Figure Legend Snippet: Time course of active TGF-β generation in incisional wound repair. Wound tissue, surrounding tissue, and normal skin were embedded in methyl cellulose. Four cryosections (20 μm) representing four wounds of one animal were prepared on duplicate coverslips and incubated overnight with MLECs. Twenty animals were used for this time course (two for each time point). MLECs were lysed and luciferase activity was determined and converted to TGF-β concentration (pg/mm 3 ) using a recombinant human TGF-β1 standard curve. The data represent mean values ± SEM of eight determinations in duplicate.

    Techniques Used: Incubation, Luciferase, Activity Assay, Concentration Assay, Recombinant

    PAI/L assay of cryosections. MLECs were plated for 4 hours into 24-well plates (3 × 10 5 /ml, 500 μl/well) and incubated overnight with coverslips carrying four cryosections each. The coverslips were placed upside down onto the cells ( A ). To generate a TGF-β standard curve, MLECs were incubated with serial dilutions of recombinant human TGF-β1 and coverslips coated with methyl cellulose ( B ). Methyl cellulose used for tissue embedding did not significantly affect the PAI/L assay.
    Figure Legend Snippet: PAI/L assay of cryosections. MLECs were plated for 4 hours into 24-well plates (3 × 10 5 /ml, 500 μl/well) and incubated overnight with coverslips carrying four cryosections each. The coverslips were placed upside down onto the cells ( A ). To generate a TGF-β standard curve, MLECs were incubated with serial dilutions of recombinant human TGF-β1 and coverslips coated with methyl cellulose ( B ). Methyl cellulose used for tissue embedding did not significantly affect the PAI/L assay.

    Techniques Used: Incubation, Recombinant

    31) Product Images from "TGF-β1 Alters Esophageal Epithelial Barrier Function by Attenuation of Claudin-7 in Eosinophilic Esophagitis"

    Article Title: TGF-β1 Alters Esophageal Epithelial Barrier Function by Attenuation of Claudin-7 in Eosinophilic Esophagitis

    Journal: Mucosal immunology

    doi: 10.1038/mi.2017.72

    TGF-β1 attenuates expression of Claudin-7 (A) Claudin mRNA expression in immortalized human esophageal epithelial (EPC2-hTERT) cells as mean fold change relative to CLDN1. CLDN 1, 4 and 7 were the predominantly expressed claudin mRNAs in the esophageal epithelium differentiated and stratified in 3D-ALI culture. (B) Transforming growth factor-β1 (TGF-β1) exposure (10 ng/ml) led to no significant effect on expression of selected epithelial barrier molecules, but decreased expression of CLDN7 mRNA and (C) claudin-7 protein in EPC2-hTERT cells at 3D-ALI (N=3, *p≤0.05, **p≤0.01). (C) Representative western blot demonstrates decreased claudin-7 protein in the presence of TGF-β1 (10 ng/ml). (E) EPC2-hTERT cells exposed to TGF-β1 (10 ng/ml) leads to attenuation in claudin-7, however when exposed to TGF-β type 1 receptor inhibitor (TGF-β RI) (5μm) prior to TGF-β1, cells were protected from the attenuation in claudin-7, indicating that the TGF-β1 pathway directly influences the attenuation of claudin-7 (N=4, ****p≤0.0001).
    Figure Legend Snippet: TGF-β1 attenuates expression of Claudin-7 (A) Claudin mRNA expression in immortalized human esophageal epithelial (EPC2-hTERT) cells as mean fold change relative to CLDN1. CLDN 1, 4 and 7 were the predominantly expressed claudin mRNAs in the esophageal epithelium differentiated and stratified in 3D-ALI culture. (B) Transforming growth factor-β1 (TGF-β1) exposure (10 ng/ml) led to no significant effect on expression of selected epithelial barrier molecules, but decreased expression of CLDN7 mRNA and (C) claudin-7 protein in EPC2-hTERT cells at 3D-ALI (N=3, *p≤0.05, **p≤0.01). (C) Representative western blot demonstrates decreased claudin-7 protein in the presence of TGF-β1 (10 ng/ml). (E) EPC2-hTERT cells exposed to TGF-β1 (10 ng/ml) leads to attenuation in claudin-7, however when exposed to TGF-β type 1 receptor inhibitor (TGF-β RI) (5μm) prior to TGF-β1, cells were protected from the attenuation in claudin-7, indicating that the TGF-β1 pathway directly influences the attenuation of claudin-7 (N=4, ****p≤0.0001).

    Techniques Used: Expressing, Western Blot

    Claudin-7 knockdown results in diminished esophageal epithelial barrier function Knockdown of CLDN7 diminished barrier function as measured by (A) transepithelial electrical resistance (TEER) and increased permeability as measured by (B) 3kDa FITC dextran paracellular flux (FITC flux) (N=11, *p≤0.05, **p≤0.01). (C) H E stained sections from CLDN7 knockdown cells grown in 3-dimensional air liquid interface (3D-ALI) culture. Arrows indicate prominent cellular separation. Black scale bar represents 50 μm. These data suggest that in the setting of attenuated claudin-7, epithelial barrier defects exist. (D) CLDN7 overexpressing cells (pLKO.1-Puro claudin-7 ORF) were generated and functional assessments were done in the presence and absence of TGF-β1 (10 ng/ml) in the 3-D ALI model. There was no significant decrease in TEER and (E) no significant increase in paracellular flux following TGF-β1 exposure. (N=5, n.s.). (F) H E stained sections from CLDN7 overexpressing cells grown in 3D-ALI culture in the absence (top panel) and presence of TGF-β1 (bottom panel). Black scale bar represents 50 μm.
    Figure Legend Snippet: Claudin-7 knockdown results in diminished esophageal epithelial barrier function Knockdown of CLDN7 diminished barrier function as measured by (A) transepithelial electrical resistance (TEER) and increased permeability as measured by (B) 3kDa FITC dextran paracellular flux (FITC flux) (N=11, *p≤0.05, **p≤0.01). (C) H E stained sections from CLDN7 knockdown cells grown in 3-dimensional air liquid interface (3D-ALI) culture. Arrows indicate prominent cellular separation. Black scale bar represents 50 μm. These data suggest that in the setting of attenuated claudin-7, epithelial barrier defects exist. (D) CLDN7 overexpressing cells (pLKO.1-Puro claudin-7 ORF) were generated and functional assessments were done in the presence and absence of TGF-β1 (10 ng/ml) in the 3-D ALI model. There was no significant decrease in TEER and (E) no significant increase in paracellular flux following TGF-β1 exposure. (N=5, n.s.). (F) H E stained sections from CLDN7 overexpressing cells grown in 3D-ALI culture in the absence (top panel) and presence of TGF-β1 (bottom panel). Black scale bar represents 50 μm.

    Techniques Used: Permeability, Staining, Generated, Functional Assay

    TGF-β1 diminishes esophageal epithelial barrier Using immortalized human esophageal epithelial (EPC2-TERT) cells in a stratified squamous 3-dimensional air liquid interface (3D-ALI) culture, transforming growth factor-β1 (TGF-β1) (10 ng/ml) diminished barrier as measured by (A) transepithelial electrical resistance (TEER) and increased permeability as measured by (B) 3kDa FITC dextran paracellular flux (FITC flux) (N=4–8, *p≤0.05, **p≤0.01). (C) H E stained sections from EPC2-TERT cells grown in 3D-ALI culture and treated with TGF-β1 (10 ng/ml). Arrows indicate prominent cellular separation. Black scale bar represents 50 μm. (D) To confirm that TGF-β1 signaling is increased, we performed western blot densitometry which shows increase in protein expression of phosphorylated SMAD2/3 (pSMAD2/3) when cells are exposed to TGF-β1 (10 ng/ml), indicating that TGF-β1 activates SMAD2/3 via phosphorylation in the in vitro 3-D ALI model (N=4, *p≤0.05). (E) Representative western blot showing increase in protein expression of pSMAD2/3 in vitro when exposed to TGF-β1 (10 ng/ml). (F) Protein expression of pSMAD2/3 and total SMAD2/3 when EPC2-TERT cells exposed to TGF-β1 (10 ng/ml). Data are expressed as mean fold change versus unstimulated controls ± SEM. Treatment with recombinant human TGF-β1 (10 ng/ml) occurred at the start of 3D-ALI exposure and during the process of differentiation and stratification on days 7 and 9.
    Figure Legend Snippet: TGF-β1 diminishes esophageal epithelial barrier Using immortalized human esophageal epithelial (EPC2-TERT) cells in a stratified squamous 3-dimensional air liquid interface (3D-ALI) culture, transforming growth factor-β1 (TGF-β1) (10 ng/ml) diminished barrier as measured by (A) transepithelial electrical resistance (TEER) and increased permeability as measured by (B) 3kDa FITC dextran paracellular flux (FITC flux) (N=4–8, *p≤0.05, **p≤0.01). (C) H E stained sections from EPC2-TERT cells grown in 3D-ALI culture and treated with TGF-β1 (10 ng/ml). Arrows indicate prominent cellular separation. Black scale bar represents 50 μm. (D) To confirm that TGF-β1 signaling is increased, we performed western blot densitometry which shows increase in protein expression of phosphorylated SMAD2/3 (pSMAD2/3) when cells are exposed to TGF-β1 (10 ng/ml), indicating that TGF-β1 activates SMAD2/3 via phosphorylation in the in vitro 3-D ALI model (N=4, *p≤0.05). (E) Representative western blot showing increase in protein expression of pSMAD2/3 in vitro when exposed to TGF-β1 (10 ng/ml). (F) Protein expression of pSMAD2/3 and total SMAD2/3 when EPC2-TERT cells exposed to TGF-β1 (10 ng/ml). Data are expressed as mean fold change versus unstimulated controls ± SEM. Treatment with recombinant human TGF-β1 (10 ng/ml) occurred at the start of 3D-ALI exposure and during the process of differentiation and stratification on days 7 and 9.

    Techniques Used: Permeability, Staining, Western Blot, Expressing, In Vitro, Recombinant

    32) Product Images from "Involvement of p300/CBP and epigenetic histone acetylation in TGF-?1-mediated gene transcription in mesangial cells"

    Article Title: Involvement of p300/CBP and epigenetic histone acetylation in TGF-?1-mediated gene transcription in mesangial cells

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00523.2012

    TGF-β1 enhances the interactions of Smad with the p300 and Sp1 proteins. A : nuclear lysates from RMCs treated either with TGF-β1 (5 ng/ml) for indicated time periods or control cells (treated with vehicle for 6 h) were immunoprecipitated with Smad2/3 antibody. Eluted proteins were immunoblotted with acetylated lysine or p300 antibody. B : nuclear lysates from MCs treated with vehicle control (ctrol) or with TGF-β1 (5 ng/ml) were immunoprecipitated with p300 antibody. Eluted proteins were immunoblotted with Smad2/3 or Sp1 antibody, and the input lysate was blotted with Smad2/3 antibody to normalize protein levels. Data shown are representative of 2 experiments.
    Figure Legend Snippet: TGF-β1 enhances the interactions of Smad with the p300 and Sp1 proteins. A : nuclear lysates from RMCs treated either with TGF-β1 (5 ng/ml) for indicated time periods or control cells (treated with vehicle for 6 h) were immunoprecipitated with Smad2/3 antibody. Eluted proteins were immunoblotted with acetylated lysine or p300 antibody. B : nuclear lysates from MCs treated with vehicle control (ctrol) or with TGF-β1 (5 ng/ml) were immunoprecipitated with p300 antibody. Eluted proteins were immunoblotted with Smad2/3 or Sp1 antibody, and the input lysate was blotted with Smad2/3 antibody to normalize protein levels. Data shown are representative of 2 experiments.

    Techniques Used: Immunoprecipitation

    TGF-β1 stimulation leads to an increase in CBP and p300 occupancy at the PAI-1 and p21 promoters. RMCs were treated with TGF-β1 (5 ng/ml) for 0.5, 2, or 6 h and control cells with vehicle (6 h). ChIP assays were performed to evaluate CBP ( A ) and p300 ( B ) occupancy at the PAI-1 promoter regions (P1 and P2). C and D : ChIP assays of CBP ( C ) and p300 ( D ) occupancy at the p21 promoter. E : ChIP assays of CBP occupancy at the CypA promoter. Bar graphs represent relative enrichment levels normalized to the input and expressed as fold over untreated controls (means ± SE, n = 3). * P
    Figure Legend Snippet: TGF-β1 stimulation leads to an increase in CBP and p300 occupancy at the PAI-1 and p21 promoters. RMCs were treated with TGF-β1 (5 ng/ml) for 0.5, 2, or 6 h and control cells with vehicle (6 h). ChIP assays were performed to evaluate CBP ( A ) and p300 ( B ) occupancy at the PAI-1 promoter regions (P1 and P2). C and D : ChIP assays of CBP ( C ) and p300 ( D ) occupancy at the p21 promoter. E : ChIP assays of CBP occupancy at the CypA promoter. Bar graphs represent relative enrichment levels normalized to the input and expressed as fold over untreated controls (means ± SE, n = 3). * P

    Techniques Used: Chromatin Immunoprecipitation

    HAT activities of CBP and p300, but not p/CAF, are essential for TGF-β1-induced PAI-1 and p21 gene expression. A – C : quiescent RMCs were transiently transfected with EGFP (GFP), CBP, p300, or p/CAF (pCAF) expression vectors and then treated with TGF-β1 (5 ng/ml) for 2 h. PAI-1, p21, and cyclophilin A (CypA) mRNA expression was analyzed by qRT-PCR. D and E : RMCs were transiently transfected with vectors expressing GFP, D/N mutants of CBP, p300, or p/CAF and then treated with TGF-β1 for 6 h. PAI-1 and p21 mRNA expression was analyzed by qRT-PCR. Values are means ± SE ( n = 3). * P
    Figure Legend Snippet: HAT activities of CBP and p300, but not p/CAF, are essential for TGF-β1-induced PAI-1 and p21 gene expression. A – C : quiescent RMCs were transiently transfected with EGFP (GFP), CBP, p300, or p/CAF (pCAF) expression vectors and then treated with TGF-β1 (5 ng/ml) for 2 h. PAI-1, p21, and cyclophilin A (CypA) mRNA expression was analyzed by qRT-PCR. D and E : RMCs were transiently transfected with vectors expressing GFP, D/N mutants of CBP, p300, or p/CAF and then treated with TGF-β1 for 6 h. PAI-1 and p21 mRNA expression was analyzed by qRT-PCR. Values are means ± SE ( n = 3). * P

    Techniques Used: HAT Assay, Expressing, Transfection, Quantitative RT-PCR

    High glucose (HG) induces H3K9/14Ac at the PAI-1 and p21 promoters in RMCs, and this is blocked by a TGF-β1-specific antibody. A and B : RMCs were cultured in normal glucose (NG; 5.5 mM glucose+20 mM mannitol) or HG (25 mM glucose) for 48 h or 72 h and then subjected to ChIP assays to determine H3K9/14Ac at the PAI-1 ( A ) and p21 ( B ) promoter regions. # P
    Figure Legend Snippet: High glucose (HG) induces H3K9/14Ac at the PAI-1 and p21 promoters in RMCs, and this is blocked by a TGF-β1-specific antibody. A and B : RMCs were cultured in normal glucose (NG; 5.5 mM glucose+20 mM mannitol) or HG (25 mM glucose) for 48 h or 72 h and then subjected to ChIP assays to determine H3K9/14Ac at the PAI-1 ( A ) and p21 ( B ) promoter regions. # P

    Techniques Used: Cell Culture, Chromatin Immunoprecipitation

    Model for TGF-β1-mediated promoter recruitment of p300/CBP and histone acetylation, and their roles in gene expression in mesangial cells. TGF-β1 induces dynamic changes in histone lysine acetylation at target gene promoters such as PAI-1 and p21 through enhanced recruitment of p300/CBP in glomerular MCs. Furthermore, it also promotes interactions of p300/CBP with transcription factors such as Smad2/3 and Sp1, and acetylation of Smad2/3 by p300. These events play key roles in augmenting TGF-β1-induced gene transcription under diabetic conditions. Thus TGF-β1-induced transcriptional factor activation can cooperate with epigenetic mechanisms in chromatin to regulate pathological gene expression associated with diabetic nephropathy (DN).
    Figure Legend Snippet: Model for TGF-β1-mediated promoter recruitment of p300/CBP and histone acetylation, and their roles in gene expression in mesangial cells. TGF-β1 induces dynamic changes in histone lysine acetylation at target gene promoters such as PAI-1 and p21 through enhanced recruitment of p300/CBP in glomerular MCs. Furthermore, it also promotes interactions of p300/CBP with transcription factors such as Smad2/3 and Sp1, and acetylation of Smad2/3 by p300. These events play key roles in augmenting TGF-β1-induced gene transcription under diabetic conditions. Thus TGF-β1-induced transcriptional factor activation can cooperate with epigenetic mechanisms in chromatin to regulate pathological gene expression associated with diabetic nephropathy (DN).

    Techniques Used: Expressing, Activation Assay

    CREB binding protein (CBP) and p300, but not p300/CBP-activating factor (p/CAF), augments transforming growth factor (TGF)-β1-induced plasminogen activator inhibitor-1 (PAI-1) and p21 promoter transactivation. A and B : expression of PAI-1 ( A ) and p21 ( B ) mRNAs in rat mesangial cells (RMCs) treated with TGF-β1 relative to control (ctrol) cells. Quiescent RMCs were treated with TGF-β1 (5 ng/ml) from 0.5 to 24 h, and ctrol cells were treated with vehicle. Total RNA was prepared from TGF-β1 treated RMCs at indicated time points while ctrol cells were harvested at the 24-h time point. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR), normalized with internal control gene β-actin and results were expressed as fold over control. Values are means ± SE ( n = 3). * P
    Figure Legend Snippet: CREB binding protein (CBP) and p300, but not p300/CBP-activating factor (p/CAF), augments transforming growth factor (TGF)-β1-induced plasminogen activator inhibitor-1 (PAI-1) and p21 promoter transactivation. A and B : expression of PAI-1 ( A ) and p21 ( B ) mRNAs in rat mesangial cells (RMCs) treated with TGF-β1 relative to control (ctrol) cells. Quiescent RMCs were treated with TGF-β1 (5 ng/ml) from 0.5 to 24 h, and ctrol cells were treated with vehicle. Total RNA was prepared from TGF-β1 treated RMCs at indicated time points while ctrol cells were harvested at the 24-h time point. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR), normalized with internal control gene β-actin and results were expressed as fold over control. Values are means ± SE ( n = 3). * P

    Techniques Used: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    HDACs suppress TGF-β1-induced PAI-1 and p21 promoter transactivation and expression. A and B : RMCs were transiently cotransfected with HDAC1, HDAC5, or a control GFP expression vector along with PAI-1-luciferase ( A ) or p21-luciferase ( B ) reporter constructs and then stimulated with TGF-β1 (5 ng/ml) for 2 h. The relative luciferase activities were determined by comparison with the activity of untreated GFP. Values are means ± SE ( n = 3). # P
    Figure Legend Snippet: HDACs suppress TGF-β1-induced PAI-1 and p21 promoter transactivation and expression. A and B : RMCs were transiently cotransfected with HDAC1, HDAC5, or a control GFP expression vector along with PAI-1-luciferase ( A ) or p21-luciferase ( B ) reporter constructs and then stimulated with TGF-β1 (5 ng/ml) for 2 h. The relative luciferase activities were determined by comparison with the activity of untreated GFP. Values are means ± SE ( n = 3). # P

    Techniques Used: Expressing, Plasmid Preparation, Luciferase, Construct, Activity Assay

    TGF-β1 induces histone H3K9/14Ac at the PAI-1 and p21 promoters. A : upstream promoter regions of the PAI-I, p21, and CypA genes. Location of primers used for the chromatin immunoprecipitation (ChIP)-qPCR assays labeled P1, P2, or P3 are indicated by short arrows. RMCs were treated with TGF-β1 (5 ng/ml) for indicated time periods up to 6 h. The control samples were harvested at 6 h along with samples treated with TGF-β1 for 6 h. B – D : ChIP assays were performed with H3K9/14Ac antibody, and ChIP-enriched DNA was amplified with primers spanning the 3 regions of the PAI-1 promoter. E : ChIP analysis of H3K9/14Ac on the p21 promoter. F : ChIP analysis of H3K9/14Ac on the CypA promoter region. IgG was used as the antibody control. The bar graphs represent relative enrichment levels normalized to the input and expressed as fold over the untreated controls (means ± SE, n = 3). P, ChIP primers; TSS, transcription start site; SBE, Smad Binding Element; Sp1, Sp1 binding element. * P
    Figure Legend Snippet: TGF-β1 induces histone H3K9/14Ac at the PAI-1 and p21 promoters. A : upstream promoter regions of the PAI-I, p21, and CypA genes. Location of primers used for the chromatin immunoprecipitation (ChIP)-qPCR assays labeled P1, P2, or P3 are indicated by short arrows. RMCs were treated with TGF-β1 (5 ng/ml) for indicated time periods up to 6 h. The control samples were harvested at 6 h along with samples treated with TGF-β1 for 6 h. B – D : ChIP assays were performed with H3K9/14Ac antibody, and ChIP-enriched DNA was amplified with primers spanning the 3 regions of the PAI-1 promoter. E : ChIP analysis of H3K9/14Ac on the p21 promoter. F : ChIP analysis of H3K9/14Ac on the CypA promoter region. IgG was used as the antibody control. The bar graphs represent relative enrichment levels normalized to the input and expressed as fold over the untreated controls (means ± SE, n = 3). P, ChIP primers; TSS, transcription start site; SBE, Smad Binding Element; Sp1, Sp1 binding element. * P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Labeling, Amplification, Binding Assay

    Effect of trichostatin A (TSA) on basal and TGF-β1-induced H3K9/14Ac at the PAI-1 and p21 promoters. RMCs were pretreated with TSA (0.3 mM) or vehicle for 24 h, washed, and then treated with TGF-β1 (5 ng/ml) for 0.5 h. A : ChIP assays of H3K9/14Ac at PAI-1 promoter regions P1 and P2. B : ChIP analysis of H3K9/14Ac at the p21 promoter region. Bar graphs represent relative enrichment levels normalized to the input and are expressed as fold over untreated controls (means ± SE, n = 3). * P
    Figure Legend Snippet: Effect of trichostatin A (TSA) on basal and TGF-β1-induced H3K9/14Ac at the PAI-1 and p21 promoters. RMCs were pretreated with TSA (0.3 mM) or vehicle for 24 h, washed, and then treated with TGF-β1 (5 ng/ml) for 0.5 h. A : ChIP assays of H3K9/14Ac at PAI-1 promoter regions P1 and P2. B : ChIP analysis of H3K9/14Ac at the p21 promoter region. Bar graphs represent relative enrichment levels normalized to the input and are expressed as fold over untreated controls (means ± SE, n = 3). * P

    Techniques Used: Chromatin Immunoprecipitation

    33) Product Images from "Epigenetic Histone Methylation Modulates Fibrotic Gene Expression"

    Article Title: Epigenetic Histone Methylation Modulates Fibrotic Gene Expression

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2010060633

    Schematic representation of histone H3 lysine methylation in TGF-β1–mediated fibrotic gene expression in mesangial cells. Diabetic conditions and TGF-β1–induced expression of ECM-associated genes Col1a1, CTGF , and PAI-1
    Figure Legend Snippet: Schematic representation of histone H3 lysine methylation in TGF-β1–mediated fibrotic gene expression in mesangial cells. Diabetic conditions and TGF-β1–induced expression of ECM-associated genes Col1a1, CTGF , and PAI-1

    Techniques Used: Methylation, Expressing

    TGF-β1 increases the expression of ECM-associated genes in RMCs. (A) Serum depleted RMCs were stimulated with TGF-β1 (10 ng/ml) for various time periods (0.5 to 24 hours), and mRNA levels of ECM-associated genes ( Col1a1, CTGF , and PAI-1
    Figure Legend Snippet: TGF-β1 increases the expression of ECM-associated genes in RMCs. (A) Serum depleted RMCs were stimulated with TGF-β1 (10 ng/ml) for various time periods (0.5 to 24 hours), and mRNA levels of ECM-associated genes ( Col1a1, CTGF , and PAI-1

    Techniques Used: Expressing

    TGF-β1–specific antibody reverses HG-induced expression of ECM-associated genes and HG-induced changes in H3K9me2/3 at their promoters in RMCs. (A) mRNA levels of ECM-associated genes in RMCs. Serum-depleted RMCs were pretreated with TGF-β1–specific
    Figure Legend Snippet: TGF-β1–specific antibody reverses HG-induced expression of ECM-associated genes and HG-induced changes in H3K9me2/3 at their promoters in RMCs. (A) mRNA levels of ECM-associated genes in RMCs. Serum-depleted RMCs were pretreated with TGF-β1–specific

    Techniques Used: Expressing

    TGF-β1–specific antibody reverses HG-induced H3K4me at ECM gene promoters in RMCs. (A–C) Bar graphs showing the H3K4me1 (A), H3K4me2 (B), and H3K4me3 (C) levels at ECM and CypA gene promoters in RMCs pretreated with TGF-β1–specific
    Figure Legend Snippet: TGF-β1–specific antibody reverses HG-induced H3K4me at ECM gene promoters in RMCs. (A–C) Bar graphs showing the H3K4me1 (A), H3K4me2 (B), and H3K4me3 (C) levels at ECM and CypA gene promoters in RMCs pretreated with TGF-β1–specific

    Techniques Used:

    TGF-β1 upregulates H3K4me1/2/3 levels at ECM-associated gene promoters. H3K4me1 (A), H3K4me2 (B), and H3K4me3 (C) levels at indicated promoters in RMCs treated without (control, ctrl) or with TGF-β1 (10 ng/ml) for various time periods.
    Figure Legend Snippet: TGF-β1 upregulates H3K4me1/2/3 levels at ECM-associated gene promoters. H3K4me1 (A), H3K4me2 (B), and H3K4me3 (C) levels at indicated promoters in RMCs treated without (control, ctrl) or with TGF-β1 (10 ng/ml) for various time periods.

    Techniques Used:

    TGF-β1 decreases H3K9me2/3 levels at ECM-associated gene promoters in RMCs. (A) Map showing locations of Col1a1 , CTGF , and PAI-1 promoter primers used for ChIP-QPCRs. TSS, transcription start site; SBE, Smad binding elements. (B and C) Bar graphs
    Figure Legend Snippet: TGF-β1 decreases H3K9me2/3 levels at ECM-associated gene promoters in RMCs. (A) Map showing locations of Col1a1 , CTGF , and PAI-1 promoter primers used for ChIP-QPCRs. TSS, transcription start site; SBE, Smad binding elements. (B and C) Bar graphs

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay

    TGF-β1 enhances SET7/9 recruitment at ECM-associated gene promoters, and the TGF-β1–specific antibody reverses SET7/9 occupancy under HG conditions in RMCs. (A) SET7/9 recruitment at the indicated gene promoters in RMCs stimulated
    Figure Legend Snippet: TGF-β1 enhances SET7/9 recruitment at ECM-associated gene promoters, and the TGF-β1–specific antibody reverses SET7/9 occupancy under HG conditions in RMCs. (A) SET7/9 recruitment at the indicated gene promoters in RMCs stimulated

    Techniques Used:

    SET7/9 is involved in TGF-β1–induced regulation of ECM-associated genes in RMCs. (A) RMCs were transfected with various concentrations of SET7/9 siRNA (siSET7/9) or control (siNeg) oligonucleotidies. Forty-eight hours after transfection,
    Figure Legend Snippet: SET7/9 is involved in TGF-β1–induced regulation of ECM-associated genes in RMCs. (A) RMCs were transfected with various concentrations of SET7/9 siRNA (siSET7/9) or control (siNeg) oligonucleotidies. Forty-eight hours after transfection,

    Techniques Used: Transfection

    TGF-β1 induces the expression of SET7/9 mRNA and protein in RMCs. (A) SET7/9 mRNA expression in RMCs after TGF-β1 treatment for various time periods was analyzed by RT-QPCR, normalized to internal control β- actin gene, and expressed
    Figure Legend Snippet: TGF-β1 induces the expression of SET7/9 mRNA and protein in RMCs. (A) SET7/9 mRNA expression in RMCs after TGF-β1 treatment for various time periods was analyzed by RT-QPCR, normalized to internal control β- actin gene, and expressed

    Techniques Used: Expressing, Quantitative RT-PCR

    34) Product Images from "Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts"

    Article Title: Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/0022155415617988

    Western blot analysis of isolated normal human airway smooth muscle (hASMC) cells, untreated or transduced with CMV-GFP or CMV-Wnt7B adenovirus for 30 hr, then treated with or without 10 ng/ml TGF-β1 for 36 hr. Wnt7B overexpression (CMV-Wnt7B)
    Figure Legend Snippet: Western blot analysis of isolated normal human airway smooth muscle (hASMC) cells, untreated or transduced with CMV-GFP or CMV-Wnt7B adenovirus for 30 hr, then treated with or without 10 ng/ml TGF-β1 for 36 hr. Wnt7B overexpression (CMV-Wnt7B)

    Techniques Used: Western Blot, Isolation, Transduction, Over Expression

    Western blot analysis of isolated normal human lung fibroblasts, untreated or transduced with CMV-GFP or CMV-Wnt7B adenovirus for 30 hr, then treated with or without 5 ng/ml TGF-β1 for 24 hr. Wnt7B overexpression (CMV-Wnt7B) and TGF-β1,
    Figure Legend Snippet: Western blot analysis of isolated normal human lung fibroblasts, untreated or transduced with CMV-GFP or CMV-Wnt7B adenovirus for 30 hr, then treated with or without 5 ng/ml TGF-β1 for 24 hr. Wnt7B overexpression (CMV-Wnt7B) and TGF-β1,

    Techniques Used: Western Blot, Isolation, Transduction, Over Expression

    Western blot analysis of day 5-isolated normal human alveolar type 2 (hAT2) cells, untreated or transduced with CMV-LacZ or CMV-Wnt7B adenovirus (20 MOI) for 30 hr, then treated with or without 10 ng/ml TGF-β1 for 24 hr. TGF-β1 alone had
    Figure Legend Snippet: Western blot analysis of day 5-isolated normal human alveolar type 2 (hAT2) cells, untreated or transduced with CMV-LacZ or CMV-Wnt7B adenovirus (20 MOI) for 30 hr, then treated with or without 10 ng/ml TGF-β1 for 24 hr. TGF-β1 alone had

    Techniques Used: Western Blot, Isolation, Transduction

    Wnt5A expression in shWnt7B-silenced normal human lung fibroblasts (hLFs) or overexpressing Wnt7B when TGF-β1 signaling is inhibited. Normal hLFs were adenovirally transduced to silence Wnt7B (shWnt7B) or with silencing Control (shScramble) before
    Figure Legend Snippet: Wnt5A expression in shWnt7B-silenced normal human lung fibroblasts (hLFs) or overexpressing Wnt7B when TGF-β1 signaling is inhibited. Normal hLFs were adenovirally transduced to silence Wnt7B (shWnt7B) or with silencing Control (shScramble) before

    Techniques Used: Expressing

    Wnt5A expression in shWnt7B-silenced IPF fibroblasts/myofibroblasts (IPFFs) or overexpressing Wnt7B when TGF-β1 signaling is inhibited. IPFF cells were transduced and treated as in . (A) Western blot. Silenced IPFF cells not transduced
    Figure Legend Snippet: Wnt5A expression in shWnt7B-silenced IPF fibroblasts/myofibroblasts (IPFFs) or overexpressing Wnt7B when TGF-β1 signaling is inhibited. IPFF cells were transduced and treated as in . (A) Western blot. Silenced IPFF cells not transduced

    Techniques Used: Expressing, Western Blot

    35) Product Images from "TGF-?1 + EGF-Initiated Invasive Potential in Transformed Human Keratinocytes Is Coupled to a Plasmin/MMP-10/MMP-1-Dependent Collagen Remodeling Axis: Role for PAI-1"

    Article Title: TGF-?1 + EGF-Initiated Invasive Potential in Transformed Human Keratinocytes Is Coupled to a Plasmin/MMP-10/MMP-1-Dependent Collagen Remodeling Axis: Role for PAI-1

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-09-0043

    MMP-10 and MMP-1 direct plasmin-dependent collagen gel dissolution. A, Western blot analysis to detect levels of active MMP-10 or MMP-1 (bottom band of each doublet) in conditioned medium from HaCaT II-4 cells cultured on collagen ± TGF-β1 + EGF for 24 h and incubated ± plasminogen (5 μg/mL) for 2–24 h. B, HaCaT II-4 cells cultured on collagen gels and stimulated with TGF-β1 + EGF for 24 h were treated ± plasminogen (20 μg/mL) ± neutralizing antibodies to MMP-1 or MMP-10 for 8 h. IgG serves as control for nonspecific antibody-based effects. Bar , 100 μm. C, Western blot analysis of conditioned medium from HaCaT II-4 cells cultured on collagen, stimulated with TGF-β1 + EGF (24 h), followed by plasminogen treatment (5 μg/mL) for the indicated times ± a neutralizing antibody to MMP-10 (30 μg/mL). Extracellular signal-regulated kinase ( ERK ) represents a loading control for each blot. Representative of multiple experiments. In all experiments, TGF-β1 is 1 ng/mL and EGF is 10 ng/mL.
    Figure Legend Snippet: MMP-10 and MMP-1 direct plasmin-dependent collagen gel dissolution. A, Western blot analysis to detect levels of active MMP-10 or MMP-1 (bottom band of each doublet) in conditioned medium from HaCaT II-4 cells cultured on collagen ± TGF-β1 + EGF for 24 h and incubated ± plasminogen (5 μg/mL) for 2–24 h. B, HaCaT II-4 cells cultured on collagen gels and stimulated with TGF-β1 + EGF for 24 h were treated ± plasminogen (20 μg/mL) ± neutralizing antibodies to MMP-1 or MMP-10 for 8 h. IgG serves as control for nonspecific antibody-based effects. Bar , 100 μm. C, Western blot analysis of conditioned medium from HaCaT II-4 cells cultured on collagen, stimulated with TGF-β1 + EGF (24 h), followed by plasminogen treatment (5 μg/mL) for the indicated times ± a neutralizing antibody to MMP-10 (30 μg/mL). Extracellular signal-regulated kinase ( ERK ) represents a loading control for each blot. Representative of multiple experiments. In all experiments, TGF-β1 is 1 ng/mL and EGF is 10 ng/mL.

    Techniques Used: Western Blot, Cell Culture, Incubation

    TGF-β1 + EGF stimulation enhances MMP-dependent collagen gel remodeling and invasion by HaCaT II-4 cells. A, OptiCell tissue culture chambers were used to visualize collagen gel invasion 6 d post-stimulation with TGF-β1 and/or EGF. The diagram illustrates the chamber setup and serves as an orientation tool. Two magnification levels of the same chamber are presented. Bar, 100 μm. B, modified Boyden chamber assays were used to quantify invasion following TGF-β1 + EGF stimulation overnight. Insets, representative quantitative fields. Bar, 100 μm. Mean ± SE of multiple fields. *, P
    Figure Legend Snippet: TGF-β1 + EGF stimulation enhances MMP-dependent collagen gel remodeling and invasion by HaCaT II-4 cells. A, OptiCell tissue culture chambers were used to visualize collagen gel invasion 6 d post-stimulation with TGF-β1 and/or EGF. The diagram illustrates the chamber setup and serves as an orientation tool. Two magnification levels of the same chamber are presented. Bar, 100 μm. B, modified Boyden chamber assays were used to quantify invasion following TGF-β1 + EGF stimulation overnight. Insets, representative quantitative fields. Bar, 100 μm. Mean ± SE of multiple fields. *, P

    Techniques Used: Modification

    Plasmin inhibition blocks MMP-dependent collagen gel dissolution. A, Western blot of endogenous plasminogen from HaCaT II-4 and HepG2 cells cultured on collagen (serum-free) overnight and then stimulated ± TGF-β1 + EGF for 8 h. Two samples of conditioned medium are presented for each. Purified human plasminogen was included as a control ( Plg ). The diagram illustrates mechanisms associated with plasmin-based pericellular proteolysis and points of inhibition; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor. B, HaCaT II-4 cells cultured on collagen type 1 gels were stimulated with TGF-β1 and/or EGF overnight followed by treatment with plasminogen (20 μg/mL; 20 h). Arrowheads, areas enlarged for insets, highlighting fibrillar characteristics of intact collagen gels versus conditions following TGF-β1 + EGF + plasminogen treatment. Bar , 100 μm. C, TGF-β1 + EGF-stimulated HaCaT II-4 cells cultured on collagen gels and treated with plasminogen (20 μg/mL) ± serine protease inhibitors aprotinin or α 2 -antiplasmin (α 2 -AP ), cysteine protease inhibitor E-64, or the MMP inhibitor GM6001. Bars , 100 μm. D, quantification of collagen digestion by HaCaT II-4 cells stimulated with TGF-β1 and/or EGF (24 h) ± plasminogen (20 μg/mL) and inhibitors aprotinin (10 μmol/L) or GM6001 (20 μmol/L) was achieved by measuring the release of FITC-labeled collagen type 1 fragments into the medium 7.5 h post-plasminogen addition. Means ± SE of duplicate wells, representative of multiple experiments. *, P
    Figure Legend Snippet: Plasmin inhibition blocks MMP-dependent collagen gel dissolution. A, Western blot of endogenous plasminogen from HaCaT II-4 and HepG2 cells cultured on collagen (serum-free) overnight and then stimulated ± TGF-β1 + EGF for 8 h. Two samples of conditioned medium are presented for each. Purified human plasminogen was included as a control ( Plg ). The diagram illustrates mechanisms associated with plasmin-based pericellular proteolysis and points of inhibition; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor. B, HaCaT II-4 cells cultured on collagen type 1 gels were stimulated with TGF-β1 and/or EGF overnight followed by treatment with plasminogen (20 μg/mL; 20 h). Arrowheads, areas enlarged for insets, highlighting fibrillar characteristics of intact collagen gels versus conditions following TGF-β1 + EGF + plasminogen treatment. Bar , 100 μm. C, TGF-β1 + EGF-stimulated HaCaT II-4 cells cultured on collagen gels and treated with plasminogen (20 μg/mL) ± serine protease inhibitors aprotinin or α 2 -antiplasmin (α 2 -AP ), cysteine protease inhibitor E-64, or the MMP inhibitor GM6001. Bars , 100 μm. D, quantification of collagen digestion by HaCaT II-4 cells stimulated with TGF-β1 and/or EGF (24 h) ± plasminogen (20 μg/mL) and inhibitors aprotinin (10 μmol/L) or GM6001 (20 μmol/L) was achieved by measuring the release of FITC-labeled collagen type 1 fragments into the medium 7.5 h post-plasminogen addition. Means ± SE of duplicate wells, representative of multiple experiments. *, P

    Techniques Used: Inhibition, Western Blot, Cell Culture, Purification, Protease Inhibitor, Labeling

    TGF-β1 + EGF stimulation increases MMP-10 levels in HaCaT II-4 cells cultured on collagen. A, evaluation of downstream MMP targets by protein microarray analysis of conditioned medium from HaCaT II-4 cells cultured on collagen gels and stimulated with TGF-β1 + EGF for 30 h. B, immunofluorescence microscopy of HaCaT II-4 cells cultured on collagen-coated coverslips (50 μg/mL) and stimulated with TGF-β1 and/or EGF (60 h). Green, MMP-10; cytoskeletal actin ( red ) was utilized for cell morphology. Bar , 10 μm. In all experiments, TGF-β1 is 1 ng/mL and EGF is 10 ng/mL.
    Figure Legend Snippet: TGF-β1 + EGF stimulation increases MMP-10 levels in HaCaT II-4 cells cultured on collagen. A, evaluation of downstream MMP targets by protein microarray analysis of conditioned medium from HaCaT II-4 cells cultured on collagen gels and stimulated with TGF-β1 + EGF for 30 h. B, immunofluorescence microscopy of HaCaT II-4 cells cultured on collagen-coated coverslips (50 μg/mL) and stimulated with TGF-β1 and/or EGF (60 h). Green, MMP-10; cytoskeletal actin ( red ) was utilized for cell morphology. Bar , 10 μm. In all experiments, TGF-β1 is 1 ng/mL and EGF is 10 ng/mL.

    Techniques Used: Cell Culture, Microarray, Immunofluorescence, Microscopy

    PAI-1 regulates MMP-10/MMP-1–dependent collagen gel dissolution by HaCaT II-4 cells. A, cell-based ELISA for PAI-1 in TGF-β1 and/or EGF-stimulated HaCaT II-4 cells cultured on collagen-coated tissue culture plastic (50 μg/mL). AG1478 was added 30 min before stimulation with TGF-β1 and/or EGF (15 h). PAI-1 was detected by colorimetric assay (see Materials and Methods). *, P
    Figure Legend Snippet: PAI-1 regulates MMP-10/MMP-1–dependent collagen gel dissolution by HaCaT II-4 cells. A, cell-based ELISA for PAI-1 in TGF-β1 and/or EGF-stimulated HaCaT II-4 cells cultured on collagen-coated tissue culture plastic (50 μg/mL). AG1478 was added 30 min before stimulation with TGF-β1 and/or EGF (15 h). PAI-1 was detected by colorimetric assay (see Materials and Methods). *, P

    Techniques Used: In-Cell ELISA, Cell Culture, Colorimetric Assay

    TGF-β1 + EGF costimulation induces EMT-like plasticity in HaCaT II-4 cells cultured on a three-dimensional collagen matrix. A and B, immunocytochemical detection of EMT-associated processes in TGF-β1 and/or EGF stimulated HaCaT II-4 cells cultured on either a thin collagen coat ( A ) or a three-dimensional collagen gel ( B ). Cells were stimulated 48 h (tubulin) and 60 h (vimentin, E-cadherin, and N-cadherin) with TGF-β1 and/or EGF. Green, vimentin, N-cadherin, and E-cadherin; red, cytoskeletal actin; yellow, colocalization between these two fluorochromes. Tubulin labeling ( A and B ) illustrates morphologic differences for each condition. Bar , 100 μm ( A ) or 50 μm ( B ). Bar , 50 μm for vimentin, N-cadherin, and E-cadherin ( A and B ). B, enlarged insets show the absence or presence of vimentin, E-cadherin, or N-cadherin. C, Western blot analysis of N-cadherin and E-cadherin from HaCaT II-4 cells cultured on collagen gels and stimulated with TGF-β1 and/or EGF for 48 h. (0), nonstimulated cells. In all experiments, TGF-β1 is 1 ng/mL and EGF is 10 ng/mL.
    Figure Legend Snippet: TGF-β1 + EGF costimulation induces EMT-like plasticity in HaCaT II-4 cells cultured on a three-dimensional collagen matrix. A and B, immunocytochemical detection of EMT-associated processes in TGF-β1 and/or EGF stimulated HaCaT II-4 cells cultured on either a thin collagen coat ( A ) or a three-dimensional collagen gel ( B ). Cells were stimulated 48 h (tubulin) and 60 h (vimentin, E-cadherin, and N-cadherin) with TGF-β1 and/or EGF. Green, vimentin, N-cadherin, and E-cadherin; red, cytoskeletal actin; yellow, colocalization between these two fluorochromes. Tubulin labeling ( A and B ) illustrates morphologic differences for each condition. Bar , 100 μm ( A ) or 50 μm ( B ). Bar , 50 μm for vimentin, N-cadherin, and E-cadherin ( A and B ). B, enlarged insets show the absence or presence of vimentin, E-cadherin, or N-cadherin. C, Western blot analysis of N-cadherin and E-cadherin from HaCaT II-4 cells cultured on collagen gels and stimulated with TGF-β1 and/or EGF for 48 h. (0), nonstimulated cells. In all experiments, TGF-β1 is 1 ng/mL and EGF is 10 ng/mL.

    Techniques Used: Cell Culture, Labeling, Western Blot

    36) Product Images from "Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components"

    Article Title: Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components

    Journal: Cytotherapy

    doi: 10.3109/14653249.2012.655420

    Multiplex analysis of human PL. (A) Cytokine concentrations of different PL-PPC preparations in pg/mL. Number of analyzed batches: n = 3 for G-CSF, GM-CSF, INF-γ, TNF-α, IL-lα, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, VEGF, MIP-lα, MIP-1β, GRO (CXCLl/2/3), TGF-β1 and bFGF; and n = 9 for sCD40L, sVCAM-1, sICAM-1, PDGF-AA, PDGF-AB/BB and RANTES. (B) Comparing cytokine content of PL-PPC and PL-APC. Number of analyzed batches: n = 3 forTGF-β1 and bFGF in PL-APC and PL-PPC; n = 4 for sCD40L, sVCAM-1, sICAM-1, PDGF-AA, PDGF-AB/BB and RANTES in PL-APC; and n = 6 for these cytokines in PL-PPC. Open bars depict PL-PPC and filled bars PL-APC.
    Figure Legend Snippet: Multiplex analysis of human PL. (A) Cytokine concentrations of different PL-PPC preparations in pg/mL. Number of analyzed batches: n = 3 for G-CSF, GM-CSF, INF-γ, TNF-α, IL-lα, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, VEGF, MIP-lα, MIP-1β, GRO (CXCLl/2/3), TGF-β1 and bFGF; and n = 9 for sCD40L, sVCAM-1, sICAM-1, PDGF-AA, PDGF-AB/BB and RANTES. (B) Comparing cytokine content of PL-PPC and PL-APC. Number of analyzed batches: n = 3 forTGF-β1 and bFGF in PL-APC and PL-PPC; n = 4 for sCD40L, sVCAM-1, sICAM-1, PDGF-AA, PDGF-AB/BB and RANTES in PL-APC; and n = 6 for these cytokines in PL-PPC. Open bars depict PL-PPC and filled bars PL-APC.

    Techniques Used: Multiplex Assay

    Inhibition of PL components bFGF, PDGF-BB and TGF-β1 by neutralizing antibodies as single factors or in combination significantly reduces MSC proliferation. Data are presented as mean and SD values of quadruplicate wells of three independent experiments. Statistical significance of the effect of inhibition of bFGF, TGF-β1 and/or PDGF-BB on MSC proliferation: * P
    Figure Legend Snippet: Inhibition of PL components bFGF, PDGF-BB and TGF-β1 by neutralizing antibodies as single factors or in combination significantly reduces MSC proliferation. Data are presented as mean and SD values of quadruplicate wells of three independent experiments. Statistical significance of the effect of inhibition of bFGF, TGF-β1 and/or PDGF-BB on MSC proliferation: * P

    Techniques Used: Inhibition

    37) Product Images from "Tumor-infiltrating myeloid cells activate Dll4/Notch/TGF-β signaling to drive malignant progression"

    Article Title: Tumor-infiltrating myeloid cells activate Dll4/Notch/TGF-β signaling to drive malignant progression

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-13-3118

    Dll4/Notch and TGF-β signaling cooperate to promote LLC1 cell growth. (A,B) LLC1 (A) and EL4 (B) proliferation to TGF-β1 with or without the inhibitors DAPT or DBZ; representative of 5 experiments. Data are cpm averages ± SD of triplicate cultures. (C) Modulation of SMADs phosphorylation and cMyc protein levels in tumor cells by TGF-β1 with or without DAPT. Representative results; band intensity ratios relative to actin are shown. (D) Relative gene expression in tumor cells cultured with TGF-β1 (2ng/ml/48 hr) alone or with DAPT (1µM). Data are averages ± SD; n=4–5 replicates; *p
    Figure Legend Snippet: Dll4/Notch and TGF-β signaling cooperate to promote LLC1 cell growth. (A,B) LLC1 (A) and EL4 (B) proliferation to TGF-β1 with or without the inhibitors DAPT or DBZ; representative of 5 experiments. Data are cpm averages ± SD of triplicate cultures. (C) Modulation of SMADs phosphorylation and cMyc protein levels in tumor cells by TGF-β1 with or without DAPT. Representative results; band intensity ratios relative to actin are shown. (D) Relative gene expression in tumor cells cultured with TGF-β1 (2ng/ml/48 hr) alone or with DAPT (1µM). Data are averages ± SD; n=4–5 replicates; *p

    Techniques Used: Expressing, Cell Culture

    38) Product Images from "Protective Role of Estrogen-induced miRNA-29 Expression in Carbon Tetrachloride-induced Mouse Liver Injury *"

    Article Title: Protective Role of Estrogen-induced miRNA-29 Expression in Carbon Tetrachloride-induced Mouse Liver Injury *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.314922

    Alterations of miR-29a and miR-29b expression levels in cells from the murine hepatoma IAR20 cells ( A and B ) and primarily cultured normal mouse hepatocytes ( C and D ) stimulated with E 2 or TGF-β1. A and C, enhancement of miR-29a and miR-29b expression
    Figure Legend Snippet: Alterations of miR-29a and miR-29b expression levels in cells from the murine hepatoma IAR20 cells ( A and B ) and primarily cultured normal mouse hepatocytes ( C and D ) stimulated with E 2 or TGF-β1. A and C, enhancement of miR-29a and miR-29b expression

    Techniques Used: Expressing, Cell Culture

    E 2 treatment prevents and TGF-β1 induces NF-κB protein nuclear accumulation in IAR20 cells. Western blot analysis of NF-κB p65 and histone H1 protein levels was performed using nuclear extracts from cells treated with 100 n m E
    Figure Legend Snippet: E 2 treatment prevents and TGF-β1 induces NF-κB protein nuclear accumulation in IAR20 cells. Western blot analysis of NF-κB p65 and histone H1 protein levels was performed using nuclear extracts from cells treated with 100 n m E

    Techniques Used: Western Blot

    Reduction of CCl 4 -induced α-SMA and collagen I expression levels in male mice by intravenous injection of Ad-miR-29a/b. A, the mRNA levels of α-SMA, collagen I, and TGF-β1 in mice treated with or without CCl 4 or Ad-miR-29a/b.
    Figure Legend Snippet: Reduction of CCl 4 -induced α-SMA and collagen I expression levels in male mice by intravenous injection of Ad-miR-29a/b. A, the mRNA levels of α-SMA, collagen I, and TGF-β1 in mice treated with or without CCl 4 or Ad-miR-29a/b.

    Techniques Used: Expressing, Mouse Assay, Injection

    39) Product Images from "SMAD3-dependent Signaling Underlies the TGF-β1-mediated Enhancement in Astrocytic iNOS Expression"

    Article Title: SMAD3-dependent Signaling Underlies the TGF-β1-mediated Enhancement in Astrocytic iNOS Expression

    Journal: Glia

    doi: 10.1002/glia.21005

    Immunocytochemical assessment of astrocytes exhibiting Smad3 nuclear accumulation and iNOS expression Cultures were treated with either vehicle [(-) TGF-β1] or TGF-β1 [(+) TGF-β1; 3ng/ml] for 24 hr prior to the addition of medium containing LPS plus IFNγ (final = 2μg/ml and 3ng/ml, respectively). Eight hr later, cultures were fixed and immunolabeled for SMAD3 (green) and iNOS (red) followed by DAPI counterstaining (blue) to illustrate the number of nuclei per field. Representative photomicrographs (63× magnification) from at least three experiments are shown for each treatment. Scale bar = 20μm.
    Figure Legend Snippet: Immunocytochemical assessment of astrocytes exhibiting Smad3 nuclear accumulation and iNOS expression Cultures were treated with either vehicle [(-) TGF-β1] or TGF-β1 [(+) TGF-β1; 3ng/ml] for 24 hr prior to the addition of medium containing LPS plus IFNγ (final = 2μg/ml and 3ng/ml, respectively). Eight hr later, cultures were fixed and immunolabeled for SMAD3 (green) and iNOS (red) followed by DAPI counterstaining (blue) to illustrate the number of nuclei per field. Representative photomicrographs (63× magnification) from at least three experiments are shown for each treatment. Scale bar = 20μm.

    Techniques Used: Expressing, Immunolabeling

    Effect of TGF-β1 in Smad3 null versus wildtype-derived astrocytes Astrocyte cultures derived from either Smad3 null (-/-) or wildtype (+/+) littermates were treated with vehicle [(-) TGF-β1] or TGF-β1 [(+) TGF-β1; 3ng/ml] for 24 hr prior to the addition of 2μg/ml LPS plus 3ng/ml IFNγ. (A) Twelve-14 hr later, culture supernatants were collected and NO production (mean μM nitrite accumulation/mg protein + SEM) was assessed (n = 15-18 from 3-4 separate dissections). (*) indicates a significant within group increase, whereas (#) indicates a significant between group diminution as determined by two-way ANOVA followed by the Bonferroni's post-hoc test. (B) Thereafter, cultures were fixed and immunolabeled for iNOS followed by DAPI counterstaining. The percentage of cells expressing iNOS per culture well was calculated and data plotted as mean % iNOS positive cells + SEM (n = 6 from 3 separate dissections). Following transformation of the percentage data, significance was assessed as in (A).
    Figure Legend Snippet: Effect of TGF-β1 in Smad3 null versus wildtype-derived astrocytes Astrocyte cultures derived from either Smad3 null (-/-) or wildtype (+/+) littermates were treated with vehicle [(-) TGF-β1] or TGF-β1 [(+) TGF-β1; 3ng/ml] for 24 hr prior to the addition of 2μg/ml LPS plus 3ng/ml IFNγ. (A) Twelve-14 hr later, culture supernatants were collected and NO production (mean μM nitrite accumulation/mg protein + SEM) was assessed (n = 15-18 from 3-4 separate dissections). (*) indicates a significant within group increase, whereas (#) indicates a significant between group diminution as determined by two-way ANOVA followed by the Bonferroni's post-hoc test. (B) Thereafter, cultures were fixed and immunolabeled for iNOS followed by DAPI counterstaining. The percentage of cells expressing iNOS per culture well was calculated and data plotted as mean % iNOS positive cells + SEM (n = 6 from 3 separate dissections). Following transformation of the percentage data, significance was assessed as in (A).

    Techniques Used: Derivative Assay, Immunolabeling, Expressing, Transformation Assay

    Effect of SB431542 following TGF-β1 addition on NO production Cultures were exposed to either vehicle [(-) TGF-β1] or TGF-β1 [(+) TGF-β1; 3ng/ml] followed by the addition of SB431542 (20μM) at the indicated times. At 24 hr (arrow), medium containing LPS plus IFNγ (final = 2μg/ml and 3ng/ml, respectively) was added to cultures. Twenty-four hr later, culture supernatants were collected and NO production (mean μM nitrite accumulation + SEM) was assessed (n = 6 from 3 separate dissections). (*) indicates a significant increase due to TGF-β1, whereas (#) indicates a significant within group SB431542-mediated diminution as compared to CON. Significance, assessed at p
    Figure Legend Snippet: Effect of SB431542 following TGF-β1 addition on NO production Cultures were exposed to either vehicle [(-) TGF-β1] or TGF-β1 [(+) TGF-β1; 3ng/ml] followed by the addition of SB431542 (20μM) at the indicated times. At 24 hr (arrow), medium containing LPS plus IFNγ (final = 2μg/ml and 3ng/ml, respectively) was added to cultures. Twenty-four hr later, culture supernatants were collected and NO production (mean μM nitrite accumulation + SEM) was assessed (n = 6 from 3 separate dissections). (*) indicates a significant increase due to TGF-β1, whereas (#) indicates a significant within group SB431542-mediated diminution as compared to CON. Significance, assessed at p

    Techniques Used:

    Effect of ALK5 inhibition on astrocytic iNOS-derived NO production Primary astrocytes were treated with the indicated concentrations of either SB431542 (A) or SB525334 (B) for 1 hr prior to addition of medium containing vehicle or TGF-β1 (final = 3ng/ml). Twenty-four hr later, cultures were cultures were spiked with medium alone or that containing LPS plus IFNγ (final = 2μg/ml and 3ng/ml, respectively). (A) Twenty-four hr or (B) 29-32hr later, cell culture supernatants were collected and NO production (mean μM nitrite accumulation + SEM) was assessed. (A), n = 6 cultures from 2 separate dissections, (B) n=10-11 from 4 separate dissections. (*) indicates a significant increase over LPS plus IFNγ alone, whereas (#) indicates a significant diminution from control as determined by one-way ANOVA followed by the Student Newman Keul's post-hoc test. Significance was assessed at p
    Figure Legend Snippet: Effect of ALK5 inhibition on astrocytic iNOS-derived NO production Primary astrocytes were treated with the indicated concentrations of either SB431542 (A) or SB525334 (B) for 1 hr prior to addition of medium containing vehicle or TGF-β1 (final = 3ng/ml). Twenty-four hr later, cultures were cultures were spiked with medium alone or that containing LPS plus IFNγ (final = 2μg/ml and 3ng/ml, respectively). (A) Twenty-four hr or (B) 29-32hr later, cell culture supernatants were collected and NO production (mean μM nitrite accumulation + SEM) was assessed. (A), n = 6 cultures from 2 separate dissections, (B) n=10-11 from 4 separate dissections. (*) indicates a significant increase over LPS plus IFNγ alone, whereas (#) indicates a significant diminution from control as determined by one-way ANOVA followed by the Student Newman Keul's post-hoc test. Significance was assessed at p

    Techniques Used: Inhibition, Derivative Assay, Cell Culture

    Immunocytochemical assessment of Smad3 nuclear translocation following TGF-β1 treatment (A) Astrocyte cultures were treated with either vehicle (0 hr) or 3ng/ml TGF-β1. After the indicated treatment times, cultures were fixed and immunolabeled for Smad3 (red) followed by DAPI counterstaining (blue) to illustrate the number of nuclei per field. A representative photomicrograph (40× magnification) is shown for each treatment condition. Scale bar = 40μm. (B) The percentage of cells exhibiting Smad3 nuclear accumulation per treatment condition was calculated (from a total of ∼100 cells present /field of view) and data plotted as mean % cells with Smad3 nuclear accumulation + SEM (n = 3-4 wells from 2 separate dissections). (*) indicates a significant increase in % of cells with Smad3 nuclear accumulation compared to control (0; non-TGF-β1 treated cells) as determined by one-way ANOVA followed by the Student Newman Keul's post-hoc test following appropriate transformation of the percentage data. Significance was assessed at p
    Figure Legend Snippet: Immunocytochemical assessment of Smad3 nuclear translocation following TGF-β1 treatment (A) Astrocyte cultures were treated with either vehicle (0 hr) or 3ng/ml TGF-β1. After the indicated treatment times, cultures were fixed and immunolabeled for Smad3 (red) followed by DAPI counterstaining (blue) to illustrate the number of nuclei per field. A representative photomicrograph (40× magnification) is shown for each treatment condition. Scale bar = 40μm. (B) The percentage of cells exhibiting Smad3 nuclear accumulation per treatment condition was calculated (from a total of ∼100 cells present /field of view) and data plotted as mean % cells with Smad3 nuclear accumulation + SEM (n = 3-4 wells from 2 separate dissections). (*) indicates a significant increase in % of cells with Smad3 nuclear accumulation compared to control (0; non-TGF-β1 treated cells) as determined by one-way ANOVA followed by the Student Newman Keul's post-hoc test following appropriate transformation of the percentage data. Significance was assessed at p

    Techniques Used: Translocation Assay, Immunolabeling, Transformation Assay

    Time course of Smad3 nuclear translocation after SB431542 addition Astrocyte cultures were treated with either vehicle [(-) TGF-β1] or TGF-β1 [3ng/ml; (+) TGF-β1] for 60 min. Thereafter, cultures were either fixed (A) or exposed to vehicle [DMSO; (-) SB431542] or SB431542 [20μM; (+) SB431542] (B) . Thirty or 90 min later, cultures were fixed ( A,B ), immunolabeled for Smad3 and counterstained for DAPI. Images from the same field of view per treatment group are shown. A representative photomicrograph (63× magnification) from at least three experiments is shown for each condition. Scale bar= 40μm.
    Figure Legend Snippet: Time course of Smad3 nuclear translocation after SB431542 addition Astrocyte cultures were treated with either vehicle [(-) TGF-β1] or TGF-β1 [3ng/ml; (+) TGF-β1] for 60 min. Thereafter, cultures were either fixed (A) or exposed to vehicle [DMSO; (-) SB431542] or SB431542 [20μM; (+) SB431542] (B) . Thirty or 90 min later, cultures were fixed ( A,B ), immunolabeled for Smad3 and counterstained for DAPI. Images from the same field of view per treatment group are shown. A representative photomicrograph (63× magnification) from at least three experiments is shown for each condition. Scale bar= 40μm.

    Techniques Used: Translocation Assay, Immunolabeling

    Analysis of TGFβ receptor mRNA expression in murine primary astrocytes Astroctye cultures were exposed to TGF-β1 (3ng/ml) or its vehicle for 24 hr prior to addition of medium alone or that containing LPS + IFNγ (final concentration= 2μg/ml + 3ng/ml, respectively; 4hr). Thereafter, total RNA was isolated and TGFβRII, ALK5 and ALK1 mRNA expression was assessed via RT-PCR. Lanes 1,5, Basal; Lanes 2,6, TGF-β1; Lanes 3,7, LPS + IFNγ; Lanes 4,8, TGF-β1 + LPS +IFNγ. Positive control (+) used was mRNA isolated from brain microendothelial cultures (BMECs). Negative control (-) is H 2 O. β-actin mRNA expression was also assessed in astrocyte samples to demonstrate RNA integrity and approximate equal loading. Data from two separate experiments (lanes 1-4 and 5-8, respectively) from two separate dissections are shown.
    Figure Legend Snippet: Analysis of TGFβ receptor mRNA expression in murine primary astrocytes Astroctye cultures were exposed to TGF-β1 (3ng/ml) or its vehicle for 24 hr prior to addition of medium alone or that containing LPS + IFNγ (final concentration= 2μg/ml + 3ng/ml, respectively; 4hr). Thereafter, total RNA was isolated and TGFβRII, ALK5 and ALK1 mRNA expression was assessed via RT-PCR. Lanes 1,5, Basal; Lanes 2,6, TGF-β1; Lanes 3,7, LPS + IFNγ; Lanes 4,8, TGF-β1 + LPS +IFNγ. Positive control (+) used was mRNA isolated from brain microendothelial cultures (BMECs). Negative control (-) is H 2 O. β-actin mRNA expression was also assessed in astrocyte samples to demonstrate RNA integrity and approximate equal loading. Data from two separate experiments (lanes 1-4 and 5-8, respectively) from two separate dissections are shown.

    Techniques Used: Expressing, Concentration Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control

    40) Product Images from "Heme Oxygenase-1 Deficiency Promotes Epithelial-Mesenchymal Transition and Renal Fibrosis"

    Article Title: Heme Oxygenase-1 Deficiency Promotes Epithelial-Mesenchymal Transition and Renal Fibrosis

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2007101099

    Characterization of primary proximal tubular epithelial cell cultures derived from HO-1 +/+ and HO-1 −/− mice. (A) Staining of HO-1 +/+ and HO-1 −/− primary proximal tubular epithelial cell cultures demonstrating expression of proximal tubular epithelial cell markers, γ-glutamyltranspeptidase (GGT) and alkaline phosphatase (Alk Phos). (B) Phase-contrast images of HO-1 +/+ and HO-1 −/− proximal tubular epithelial cell cultures showing domes in confluent cells (0 h, vehicle treated) of each genotype and phenotypic changes in response to TGF-β1 (5 ng/ml) with time, as indicated. (C) Immunoblot from cell lysates from HO-1 +/+ and HO-1 −/− proximal tubular epithelial cell cultures treated with vehicle (0 h) or TGF-β1 (5 ng/ml) for the times indicated. Blots were stripped and reprobed for β-actin to confirm loading and transfer. Representative of n = 4 independent cell cultures from separate mice/genotype.
    Figure Legend Snippet: Characterization of primary proximal tubular epithelial cell cultures derived from HO-1 +/+ and HO-1 −/− mice. (A) Staining of HO-1 +/+ and HO-1 −/− primary proximal tubular epithelial cell cultures demonstrating expression of proximal tubular epithelial cell markers, γ-glutamyltranspeptidase (GGT) and alkaline phosphatase (Alk Phos). (B) Phase-contrast images of HO-1 +/+ and HO-1 −/− proximal tubular epithelial cell cultures showing domes in confluent cells (0 h, vehicle treated) of each genotype and phenotypic changes in response to TGF-β1 (5 ng/ml) with time, as indicated. (C) Immunoblot from cell lysates from HO-1 +/+ and HO-1 −/− proximal tubular epithelial cell cultures treated with vehicle (0 h) or TGF-β1 (5 ng/ml) for the times indicated. Blots were stripped and reprobed for β-actin to confirm loading and transfer. Representative of n = 4 independent cell cultures from separate mice/genotype.

    Techniques Used: Derivative Assay, Mouse Assay, Staining, Expressing

    EMT in HO-1 +/+ and HO-1 −/− primary proximal tubular epithelial cell cultures. Immunoblot analysis of cell lysates from HO-1 +/+ and HO-1 −/− primary proximal tubular epithelial cell cultures treated with vehicle (0 h) or TGF-β1 (5 ng/ml) for the times indicated. The blots were stripped and reprobed for β-actin to confirm loading and transfer. (A) E-cadherin (E-cad) immunoblot (top) and densitometry of E-cad/β-actin (arbitrary units; bottom). (B) Claudin-2 (top) and densitometry of claudin-2/β-actin (arbitrary units; bottom). (C) α-SMA (top) and densitometry of α-SMA/β-actin (arbitrary units; bottom). * P
    Figure Legend Snippet: EMT in HO-1 +/+ and HO-1 −/− primary proximal tubular epithelial cell cultures. Immunoblot analysis of cell lysates from HO-1 +/+ and HO-1 −/− primary proximal tubular epithelial cell cultures treated with vehicle (0 h) or TGF-β1 (5 ng/ml) for the times indicated. The blots were stripped and reprobed for β-actin to confirm loading and transfer. (A) E-cadherin (E-cad) immunoblot (top) and densitometry of E-cad/β-actin (arbitrary units; bottom). (B) Claudin-2 (top) and densitometry of claudin-2/β-actin (arbitrary units; bottom). (C) α-SMA (top) and densitometry of α-SMA/β-actin (arbitrary units; bottom). * P

    Techniques Used:

    Related Articles

    Enzyme-linked Immunosorbent Assay:

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    Polymerase Chain Reaction:

    Article Title: Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene
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    Incubation:

    Article Title: Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene
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    Recombinant:

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    Article Snippet: .. Anti-MMP2, anti-TIMP1, TGF-β1 ELISA kit, and recombinant human TGF-β1 were purchased from R & D Systems. .. Bleomycin was from Sigma.

    Article Title: TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells
    Article Snippet: .. Human TGF-β1 (100B) and recombinant human TGF-β1 (anti TGF-β1 antibody) (MAB240) from R & D Systems Inc., Minneapolis, MN. .. Antibodies were purchased from the following sources: phospho-Smad2/3 (Ser423/425) (sc-11769-R), Smad4 (sc-7154), ATF3 (sc-188), c-Jun (sc-1694), p53 (sc-6243) and CD-36 (sc-5523) from Santa Cruz Biotechnology, Santa Cruz, CA; Smad2 (#3103), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828)and p-c-Jun (#9261) from Cell Signaling Technology (Danvers, MA); caspase-3 (IMG-144A) from (Imgenex, San Diego, CA); Nuclear Loading Control Anti-TATA binding protein TBP antibody (ab63766) from Abcam (Cambridge, MA); HRP-conjugated secondary anti-mouse (Amersham # NXA931) or anti-rabbit antibody (Amersham #NA9340V) from GE-Biosciences.

    Article Title: Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene
    Article Snippet: .. Recombinant human TGF-β1 and BMP2 (R & D Systems) were added individually to about 25 pairs of adult worms to final concentrations of 1.0 nM and 5.0 nM , respectively, and incubation was continued for another 24 h, after which the medium was changed and worms were incubated for an additional 24 h. Total RNA was extracted from untreated or TGF-β–treated worm pairs, and cDNA was synthesized and then subjected to PCR using different specific gene primers following the optimized PCR conditions for each gene employing the aforementioned protocol. .. PCR products were separated onto 2% agarose gels, and processed and quantified as described above.

    Synthesized:

    Article Title: Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene
    Article Snippet: .. Recombinant human TGF-β1 and BMP2 (R & D Systems) were added individually to about 25 pairs of adult worms to final concentrations of 1.0 nM and 5.0 nM , respectively, and incubation was continued for another 24 h, after which the medium was changed and worms were incubated for an additional 24 h. Total RNA was extracted from untreated or TGF-β–treated worm pairs, and cDNA was synthesized and then subjected to PCR using different specific gene primers following the optimized PCR conditions for each gene employing the aforementioned protocol. .. PCR products were separated onto 2% agarose gels, and processed and quantified as described above.

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    R&D Systems human recombinant tgf β1
    Effect of IL-6 on <t>TGF-β1</t> expression and the proliferation rate in oral cancer cells (A) Effect of IL-6 on the level of TGF-β1 was examined by Western blotting. Representative blots from experiments performed in triplicate are shown. (B) Effect of IL-6 signalling on the levels of HIF-1α, TGF-β1, vimentin and MMP-9 in oral cancer cells. Representative blots from experiments performed in triplicate are shown. Ab, antibody; W, wild-type. (C) Effects of IL-6 on the proliferation rate of oral cancer cells. The same number of cells (10 4 ) were plated on day 0 and were allowed to grow in their respective cultures. The number of viable cells after incubation for 2, 4 and 6 days was counted. The relative viable cell number normalized to that under control conditions was calculated. Values are means±S.D. for three separate experiments; * P
    Human Recombinant Tgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of IL-6 on TGF-β1 expression and the proliferation rate in oral cancer cells (A) Effect of IL-6 on the level of TGF-β1 was examined by Western blotting. Representative blots from experiments performed in triplicate are shown. (B) Effect of IL-6 signalling on the levels of HIF-1α, TGF-β1, vimentin and MMP-9 in oral cancer cells. Representative blots from experiments performed in triplicate are shown. Ab, antibody; W, wild-type. (C) Effects of IL-6 on the proliferation rate of oral cancer cells. The same number of cells (10 4 ) were plated on day 0 and were allowed to grow in their respective cultures. The number of viable cells after incubation for 2, 4 and 6 days was counted. The relative viable cell number normalized to that under control conditions was calculated. Values are means±S.D. for three separate experiments; * P

    Journal: Clinical Science (London, England : 1979)

    Article Title: Significance of the TGF-?1/IL-6 axis in oral cancer

    doi: 10.1042/CS20110434

    Figure Lengend Snippet: Effect of IL-6 on TGF-β1 expression and the proliferation rate in oral cancer cells (A) Effect of IL-6 on the level of TGF-β1 was examined by Western blotting. Representative blots from experiments performed in triplicate are shown. (B) Effect of IL-6 signalling on the levels of HIF-1α, TGF-β1, vimentin and MMP-9 in oral cancer cells. Representative blots from experiments performed in triplicate are shown. Ab, antibody; W, wild-type. (C) Effects of IL-6 on the proliferation rate of oral cancer cells. The same number of cells (10 4 ) were plated on day 0 and were allowed to grow in their respective cultures. The number of viable cells after incubation for 2, 4 and 6 days was counted. The relative viable cell number normalized to that under control conditions was calculated. Values are means±S.D. for three separate experiments; * P

    Article Snippet: Human recombinant TGF-β1, the anti-TGF-β1 antibody and the anti-IL-6 antibody were obtained from R & D Systems.

    Techniques: Expressing, Western Blot, Incubation

    Effect of the levels of TGF-β1 and IL-6 in the clinical outcome of oral cancer (A) Positive immunohistochemical (IHC) staining for IL-6 was significantly linked with TGF-β1 expression in human oral cancer specimens. Representative images of a selected tumour specimen positive for TGF-β1 and IL-6 staining are shown. (B) Overall survival differences according to the positive staining of TGF-β1 and IL-6, and disease failure.

    Journal: Clinical Science (London, England : 1979)

    Article Title: Significance of the TGF-?1/IL-6 axis in oral cancer

    doi: 10.1042/CS20110434

    Figure Lengend Snippet: Effect of the levels of TGF-β1 and IL-6 in the clinical outcome of oral cancer (A) Positive immunohistochemical (IHC) staining for IL-6 was significantly linked with TGF-β1 expression in human oral cancer specimens. Representative images of a selected tumour specimen positive for TGF-β1 and IL-6 staining are shown. (B) Overall survival differences according to the positive staining of TGF-β1 and IL-6, and disease failure.

    Article Snippet: Human recombinant TGF-β1, the anti-TGF-β1 antibody and the anti-IL-6 antibody were obtained from R & D Systems.

    Techniques: Immunohistochemistry, Staining, Expressing

    Effect of TGF-β1 on radiation-induced 8-oxoG, phospho-ATM and phosho-H2AX levels (A) Influence of TGF-β1 on radiation (RT)-induced 8-oxoG and phospho-H2AX was evaluated by immunofluorescence in cells 30 min after irradiation following pre-incubation with or without the anti-TGF-β1 antibody (Ab). Representative images and quantitative results are shown. The quantification of 8-oxoG and phospho-H2AX expression was calculated by dividing the number of cells positive for the target protein by the total cell number for each condition. Values are means±S.D. for three separate experiments. (B) The influence of TGF-β1 on radiation-induced nuclear accumulation of phospho-ATM was evaluated by immunofluorescence and Western blot analysis in cells 30 min after irradiation. p-, phospho-.

    Journal: Clinical Science (London, England : 1979)

    Article Title: Significance of the TGF-?1/IL-6 axis in oral cancer

    doi: 10.1042/CS20110434

    Figure Lengend Snippet: Effect of TGF-β1 on radiation-induced 8-oxoG, phospho-ATM and phosho-H2AX levels (A) Influence of TGF-β1 on radiation (RT)-induced 8-oxoG and phospho-H2AX was evaluated by immunofluorescence in cells 30 min after irradiation following pre-incubation with or without the anti-TGF-β1 antibody (Ab). Representative images and quantitative results are shown. The quantification of 8-oxoG and phospho-H2AX expression was calculated by dividing the number of cells positive for the target protein by the total cell number for each condition. Values are means±S.D. for three separate experiments. (B) The influence of TGF-β1 on radiation-induced nuclear accumulation of phospho-ATM was evaluated by immunofluorescence and Western blot analysis in cells 30 min after irradiation. p-, phospho-.

    Article Snippet: Human recombinant TGF-β1, the anti-TGF-β1 antibody and the anti-IL-6 antibody were obtained from R & D Systems.

    Techniques: Immunofluorescence, Irradiation, Incubation, Expressing, Western Blot

    Effect of TGF-β1 in cancer cell proliferation (A) Effects of the TGF-β1–RFP-silencing vector and TGF-β1–GFP-expression vector on the level of TGF-β1 in SCC4 cells as determined by immunofluorescence. Expression of TGF-β1 in SCC4 transfectants was also examined by Western blot analysis. W or Wild, wild-type; R-V, cells with control–RFP vector; TG-, cells with the TGF-β1–RFP-silencing vector; G-V, cells with control–GFP vector; TG + , cells with the TGF-β1–GFP-expression vector. (B) Effect of TGF-β1 on the proliferation rate of cancer cells. The same number of cells (10 4 ) were plated on to each plate on day (D) 0 and were allowed to grow in their respective cultures. The number of viable cells after incubation for 2, 4 and 6 days were counted. * P

    Journal: Clinical Science (London, England : 1979)

    Article Title: Significance of the TGF-?1/IL-6 axis in oral cancer

    doi: 10.1042/CS20110434

    Figure Lengend Snippet: Effect of TGF-β1 in cancer cell proliferation (A) Effects of the TGF-β1–RFP-silencing vector and TGF-β1–GFP-expression vector on the level of TGF-β1 in SCC4 cells as determined by immunofluorescence. Expression of TGF-β1 in SCC4 transfectants was also examined by Western blot analysis. W or Wild, wild-type; R-V, cells with control–RFP vector; TG-, cells with the TGF-β1–RFP-silencing vector; G-V, cells with control–GFP vector; TG + , cells with the TGF-β1–GFP-expression vector. (B) Effect of TGF-β1 on the proliferation rate of cancer cells. The same number of cells (10 4 ) were plated on to each plate on day (D) 0 and were allowed to grow in their respective cultures. The number of viable cells after incubation for 2, 4 and 6 days were counted. * P

    Article Snippet: Human recombinant TGF-β1, the anti-TGF-β1 antibody and the anti-IL-6 antibody were obtained from R & D Systems.

    Techniques: Plasmid Preparation, Expressing, Immunofluorescence, Western Blot, Incubation

    Effect of TGF-β1 on tumour aggressiveness and EMT changes (A) The invasive capacities of oral cancer cells with or without TGF-β1 regulation were evaluated using an invasion assay in SCC25 cells (left-hand panels) and a migration assay in SCC4 cells (right-hand panels). Representative images and quantification are shown. For the invasion assay, the relative number of invading cells normalized to that under control conditions was calculated. For the migration assay, the relative ratio, normalized to the distance under control conditions at 24 h after scratching, was calculated. Results are means±S.D. for three separate experiments; * P

    Journal: Clinical Science (London, England : 1979)

    Article Title: Significance of the TGF-?1/IL-6 axis in oral cancer

    doi: 10.1042/CS20110434

    Figure Lengend Snippet: Effect of TGF-β1 on tumour aggressiveness and EMT changes (A) The invasive capacities of oral cancer cells with or without TGF-β1 regulation were evaluated using an invasion assay in SCC25 cells (left-hand panels) and a migration assay in SCC4 cells (right-hand panels). Representative images and quantification are shown. For the invasion assay, the relative number of invading cells normalized to that under control conditions was calculated. For the migration assay, the relative ratio, normalized to the distance under control conditions at 24 h after scratching, was calculated. Results are means±S.D. for three separate experiments; * P

    Article Snippet: Human recombinant TGF-β1, the anti-TGF-β1 antibody and the anti-IL-6 antibody were obtained from R & D Systems.

    Techniques: Invasion Assay, Migration

    Effect of TGF-β1 expression on IL-6 signalling (A) Effect of TGF-β1 on IL-6 signalling was evaluated by Western blotting in SCC25 and SCC4 cells. p-, phospho-. (B) Influence of TGF-β1 on the level of IL-6 in cells was examined by real-time RT–PCR. The IL-6/β-actin ratio in cancer cells normalized to cells without treatment was calculated. Values are means±S.D. for three separate experiments; * P

    Journal: Clinical Science (London, England : 1979)

    Article Title: Significance of the TGF-?1/IL-6 axis in oral cancer

    doi: 10.1042/CS20110434

    Figure Lengend Snippet: Effect of TGF-β1 expression on IL-6 signalling (A) Effect of TGF-β1 on IL-6 signalling was evaluated by Western blotting in SCC25 and SCC4 cells. p-, phospho-. (B) Influence of TGF-β1 on the level of IL-6 in cells was examined by real-time RT–PCR. The IL-6/β-actin ratio in cancer cells normalized to cells without treatment was calculated. Values are means±S.D. for three separate experiments; * P

    Article Snippet: Human recombinant TGF-β1, the anti-TGF-β1 antibody and the anti-IL-6 antibody were obtained from R & D Systems.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    Effects of TGF-β1 on the treatment response (A) Cells were treated with 0 or 6 μg/ml cisplatin in the presence or absence of the anti-TGF-β1 antibody, and the survival rate was determined at 48 h using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2 H -tetrazolium-5-carboxanilide] assay. The relative ratio normalized to that in SCC25 cells under control conditions for 48 h was calculated. (B) Cells were treated with 0 or 4 Gy in the presence or absence of the anti-TGF-β1 antibody, and the survival rate was determined at 48 h using the XTT assay. The relative ratio normalized to that in SCC25 cells under control conditions was calculated. (C) SCC4 transfectants were irradiated with 0, 2, 4 and 6 Gy, and the survival fractions were determined by measuring the colonies after irradiation and dividing by the plating efficiency. Values are means±S.D.; P

    Journal: Clinical Science (London, England : 1979)

    Article Title: Significance of the TGF-?1/IL-6 axis in oral cancer

    doi: 10.1042/CS20110434

    Figure Lengend Snippet: Effects of TGF-β1 on the treatment response (A) Cells were treated with 0 or 6 μg/ml cisplatin in the presence or absence of the anti-TGF-β1 antibody, and the survival rate was determined at 48 h using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2 H -tetrazolium-5-carboxanilide] assay. The relative ratio normalized to that in SCC25 cells under control conditions for 48 h was calculated. (B) Cells were treated with 0 or 4 Gy in the presence or absence of the anti-TGF-β1 antibody, and the survival rate was determined at 48 h using the XTT assay. The relative ratio normalized to that in SCC25 cells under control conditions was calculated. (C) SCC4 transfectants were irradiated with 0, 2, 4 and 6 Gy, and the survival fractions were determined by measuring the colonies after irradiation and dividing by the plating efficiency. Values are means±S.D.; P

    Article Snippet: Human recombinant TGF-β1, the anti-TGF-β1 antibody and the anti-IL-6 antibody were obtained from R & D Systems.

    Techniques: XTT Assay, Irradiation

    Immunohistochemical staining of human oral cancer TMA specimens (A) Representative images of TGF-β1- and IL-6-positive staining by immunohistochemistry using the Good Speed scan slide scanning platform and Image Pro Plus 6.3. Upper row, adjacent non-malignant epithelium; lower panel, oral cancer tissue. Scale bars, 100 μm. (B) Representative data analysed by Image Pro Plus 6.3 are shown for each molecular marker. Left-hand panel, original immunohistochemical images; right-hand panel, Image Pro Plus 6.3 analysis with positive events marked in red. (C) Representative images of TGF-βRI- and TGF-βRII-positive staining by immunohistochemistry using the Good Speed slide scanning platform.

    Journal: Clinical Science (London, England : 1979)

    Article Title: Significance of the TGF-?1/IL-6 axis in oral cancer

    doi: 10.1042/CS20110434

    Figure Lengend Snippet: Immunohistochemical staining of human oral cancer TMA specimens (A) Representative images of TGF-β1- and IL-6-positive staining by immunohistochemistry using the Good Speed scan slide scanning platform and Image Pro Plus 6.3. Upper row, adjacent non-malignant epithelium; lower panel, oral cancer tissue. Scale bars, 100 μm. (B) Representative data analysed by Image Pro Plus 6.3 are shown for each molecular marker. Left-hand panel, original immunohistochemical images; right-hand panel, Image Pro Plus 6.3 analysis with positive events marked in red. (C) Representative images of TGF-βRI- and TGF-βRII-positive staining by immunohistochemistry using the Good Speed slide scanning platform.

    Article Snippet: Human recombinant TGF-β1, the anti-TGF-β1 antibody and the anti-IL-6 antibody were obtained from R & D Systems.

    Techniques: Immunohistochemistry, Staining, Marker

    Schematic representation of proposed model for TGF-β1-induced activation of TAK1 in MMC. Under unstimulated conditions TAK1 interacts with TβRI through the complex formation with TAB2 and TRAF6. TGF-β1 stimulation-induced formation

    Journal: The Journal of Biological Chemistry

    Article Title: Transforming Growth Factor-? (TGF-?1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-? Receptor Kinase Activity in Mesangial Cells *

    doi: 10.1074/jbc.M109.007146

    Figure Lengend Snippet: Schematic representation of proposed model for TGF-β1-induced activation of TAK1 in MMC. Under unstimulated conditions TAK1 interacts with TβRI through the complex formation with TAB2 and TRAF6. TGF-β1 stimulation-induced formation

    Article Snippet: Recombinant human TGF-β1 was obtained from R & D Systems (Minneapolis, MN).

    Techniques: Activation Assay

    TβRI-mediated and TGF-β1-induced TAK1 phosphorylation requires its own kinase activity and TAB1. A , TAB1 induces autophosphorylation of TAK1. FLAG-TAB1 was coexpressed in MMC with WT, kinase-deficient mutant ( KD ; K63W), or phosphorylation

    Journal: The Journal of Biological Chemistry

    Article Title: Transforming Growth Factor-? (TGF-?1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-? Receptor Kinase Activity in Mesangial Cells *

    doi: 10.1074/jbc.M109.007146

    Figure Lengend Snippet: TβRI-mediated and TGF-β1-induced TAK1 phosphorylation requires its own kinase activity and TAB1. A , TAB1 induces autophosphorylation of TAK1. FLAG-TAB1 was coexpressed in MMC with WT, kinase-deficient mutant ( KD ; K63W), or phosphorylation

    Article Snippet: Recombinant human TGF-β1 was obtained from R & D Systems (Minneapolis, MN).

    Techniques: Activity Assay, Mutagenesis

    TRAF6 mediates the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation. A , knockdown of TRAF6 by siRNA inhibits TβRI-TAK1 interaction. After transfection of MMC with control siRNA (−) or siRNA specific for

    Journal: The Journal of Biological Chemistry

    Article Title: Transforming Growth Factor-? (TGF-?1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-? Receptor Kinase Activity in Mesangial Cells *

    doi: 10.1074/jbc.M109.007146

    Figure Lengend Snippet: TRAF6 mediates the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation. A , knockdown of TRAF6 by siRNA inhibits TβRI-TAK1 interaction. After transfection of MMC with control siRNA (−) or siRNA specific for

    Article Snippet: Recombinant human TGF-β1 was obtained from R & D Systems (Minneapolis, MN).

    Techniques: Activation Assay, Transfection

    Delta‐aminolevulinate dehydratase ( ALAD ) regulates transforming growth factor‐β ( TGF ‐β)‐mediated epithelial–mesenchymal transition in MCF 10A cells. (a,b) RT –quantitative PCR analysis of ALAD expression levels in MCF 10A cells treated with TGF ‐β1 at indicated concentrations (a) and time (b) compared to the control. (c) Morphology of MCF 10A cells with Control and ALAD after 7 days of treatment with TGF ‐β1 (10 ng/ mL ) in MCF 10A cells. (d) TGF ‐β signaling activity determined by luciferase report assay. (e,f) Western blot of E‐cadherin, vimentin, and ALAD expression in indicated MCF 10A cells. (g) Invasive ability of indicated MCF 10A cells. * P

    Journal: Cancer Science

    Article Title: Downregulation of delta‐aminolevulinate dehydratase is associated with poor prognosis in patients with breast cancer

    doi: 10.1111/cas.13180

    Figure Lengend Snippet: Delta‐aminolevulinate dehydratase ( ALAD ) regulates transforming growth factor‐β ( TGF ‐β)‐mediated epithelial–mesenchymal transition in MCF 10A cells. (a,b) RT –quantitative PCR analysis of ALAD expression levels in MCF 10A cells treated with TGF ‐β1 at indicated concentrations (a) and time (b) compared to the control. (c) Morphology of MCF 10A cells with Control and ALAD after 7 days of treatment with TGF ‐β1 (10 ng/ mL ) in MCF 10A cells. (d) TGF ‐β signaling activity determined by luciferase report assay. (e,f) Western blot of E‐cadherin, vimentin, and ALAD expression in indicated MCF 10A cells. (g) Invasive ability of indicated MCF 10A cells. * P

    Article Snippet: Recombinant human TGF‐β1 was purchased from R & D Systems (Redmond, WA, USA).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Luciferase, Western Blot

    Semi-Quantitative RT-PCR Analysis of SmGCP mRNA The bottom panel shows the agarose gel separation of the PCR products of SmGCP (bottom), and the constitutively transcribed control, α-tubulin (top). Panel A: Lanes are numbered and the respective stages are listed at the bottom of the panel. Panel B: Adult worm pairs (42-d-old) were left untreated (lane 1) or treated with human TGF-β1 (1 nM; lane 2) or human BMP2 (5 nM; lane 3). Top of each panel shows a bar graph representation of the relative PCR band intensities (%) of SmGCP compared to that of α-tubulin control. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).

    Journal: PLoS Pathogens

    Article Title: Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene

    doi: 10.1371/journal.ppat.0020054

    Figure Lengend Snippet: Semi-Quantitative RT-PCR Analysis of SmGCP mRNA The bottom panel shows the agarose gel separation of the PCR products of SmGCP (bottom), and the constitutively transcribed control, α-tubulin (top). Panel A: Lanes are numbered and the respective stages are listed at the bottom of the panel. Panel B: Adult worm pairs (42-d-old) were left untreated (lane 1) or treated with human TGF-β1 (1 nM; lane 2) or human BMP2 (5 nM; lane 3). Top of each panel shows a bar graph representation of the relative PCR band intensities (%) of SmGCP compared to that of α-tubulin control. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).

    Article Snippet: Recombinant human TGF-β1 and BMP2 (R & D Systems) were added individually to about 25 pairs of adult worms to final concentrations of 1.0 nM and 5.0 nM , respectively, and incubation was continued for another 24 h, after which the medium was changed and worms were incubated for an additional 24 h. Total RNA was extracted from untreated or TGF-β–treated worm pairs, and cDNA was synthesized and then subjected to PCR using different specific gene primers following the optimized PCR conditions for each gene employing the aforementioned protocol.

    Techniques: Quantitative RT-PCR, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Standard Deviation

    Silencing of TGF-β–Induced Expression of SmGCP by Knocking Down SmTβRII Expression Semi-quantitative RT-PCR analyses for transcripts of SmGCP as well as various components of schistosomal TGF-β signaling pathways in 35-d-old and 28-d-old old worm pairs, untransformed and transformed with SmTβRII-siRNA, and either left untreated or treated with TGF-β1 (1 nM). The top panel shows the agarose gel separation of the PCR products of SmTβRII (panel B), SmTβRI (panel C), SmGCP (panel D), SmSmad4 (panel E), SmSmad2 (panel F), SmSmad1 (panel G), and the constitutively transcribed control, α-tubulin (panel A). The lanes are labeled to show detailed treatment of each sample. The bar graph representation shows the percentage values of the optical densities in pixels of the PCR bands for each gene compared to the corresponding band of α-tubulin control from the same stage. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).

    Journal: PLoS Pathogens

    Article Title: Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene

    doi: 10.1371/journal.ppat.0020054

    Figure Lengend Snippet: Silencing of TGF-β–Induced Expression of SmGCP by Knocking Down SmTβRII Expression Semi-quantitative RT-PCR analyses for transcripts of SmGCP as well as various components of schistosomal TGF-β signaling pathways in 35-d-old and 28-d-old old worm pairs, untransformed and transformed with SmTβRII-siRNA, and either left untreated or treated with TGF-β1 (1 nM). The top panel shows the agarose gel separation of the PCR products of SmTβRII (panel B), SmTβRI (panel C), SmGCP (panel D), SmSmad4 (panel E), SmSmad2 (panel F), SmSmad1 (panel G), and the constitutively transcribed control, α-tubulin (panel A). The lanes are labeled to show detailed treatment of each sample. The bar graph representation shows the percentage values of the optical densities in pixels of the PCR bands for each gene compared to the corresponding band of α-tubulin control from the same stage. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).

    Article Snippet: Recombinant human TGF-β1 and BMP2 (R & D Systems) were added individually to about 25 pairs of adult worms to final concentrations of 1.0 nM and 5.0 nM , respectively, and incubation was continued for another 24 h, after which the medium was changed and worms were incubated for an additional 24 h. Total RNA was extracted from untreated or TGF-β–treated worm pairs, and cDNA was synthesized and then subjected to PCR using different specific gene primers following the optimized PCR conditions for each gene employing the aforementioned protocol.

    Techniques: Expressing, Quantitative RT-PCR, Transformation Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Labeling, Standard Deviation

    The Transduction of TGF-β Signal to SmSmad2 via Activated SmTβRII/SmTβRI Receptor Complex, In Vitro: Co-immunoprecipitation of SmSmad2-MH2 and SmSmad4 35 S-labeled, in vitro translated products of SmSmad2-MH2 (panel A) and SmSmad2-MH2/AAA (panel B) were incubated with SmSmad4 in the presence of SmTβR-I (wt) and SmTβR-II in the presence or absence of TGF-β1 (1.0 nM) or BMP2 (5.0 nM). Radiolabeled, in vitro translated products were co-precipitated with SmSmad4, using anti-SmSmad4-linker IgG and Protein A Sepharose beads (Amersham Biosciences). Background precipitation was removed by treating 35 S-labeled in vitro translated products with anti-SmSmad4-linker IgG and Protein A Sepharose beads. The pre-cleared lysates were then used in the above-described reactions. A positive control reaction (lane 3) was included, in which SmSmad2-MH2, or the AAA mutant peptide, were reacted with SmSmad4 in the presence of the active mutant form of type I receptor, SmTβR-I (Q-D). Reactions, which contain either SmSmad2-MH2 or its AAA mutant form with SmSmad4 in the presence of wild-type SmTβRI, represent the negative controls of the assay (lane 4). Immunoprecipitated products were separated by SDS-PAGE and subjected to autofluorography. Lanes are labeled to specify the input components of each reaction. In vitro translated products (20% of input) are shown (lane 1). Percentage values of precipitated reactive radiolabeled product of each reaction are shown at the bottom of each lane.

    Journal: PLoS Pathogens

    Article Title: Schistosoma mansoni TGF-? Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene

    doi: 10.1371/journal.ppat.0020054

    Figure Lengend Snippet: The Transduction of TGF-β Signal to SmSmad2 via Activated SmTβRII/SmTβRI Receptor Complex, In Vitro: Co-immunoprecipitation of SmSmad2-MH2 and SmSmad4 35 S-labeled, in vitro translated products of SmSmad2-MH2 (panel A) and SmSmad2-MH2/AAA (panel B) were incubated with SmSmad4 in the presence of SmTβR-I (wt) and SmTβR-II in the presence or absence of TGF-β1 (1.0 nM) or BMP2 (5.0 nM). Radiolabeled, in vitro translated products were co-precipitated with SmSmad4, using anti-SmSmad4-linker IgG and Protein A Sepharose beads (Amersham Biosciences). Background precipitation was removed by treating 35 S-labeled in vitro translated products with anti-SmSmad4-linker IgG and Protein A Sepharose beads. The pre-cleared lysates were then used in the above-described reactions. A positive control reaction (lane 3) was included, in which SmSmad2-MH2, or the AAA mutant peptide, were reacted with SmSmad4 in the presence of the active mutant form of type I receptor, SmTβR-I (Q-D). Reactions, which contain either SmSmad2-MH2 or its AAA mutant form with SmSmad4 in the presence of wild-type SmTβRI, represent the negative controls of the assay (lane 4). Immunoprecipitated products were separated by SDS-PAGE and subjected to autofluorography. Lanes are labeled to specify the input components of each reaction. In vitro translated products (20% of input) are shown (lane 1). Percentage values of precipitated reactive radiolabeled product of each reaction are shown at the bottom of each lane.

    Article Snippet: Recombinant human TGF-β1 and BMP2 (R & D Systems) were added individually to about 25 pairs of adult worms to final concentrations of 1.0 nM and 5.0 nM , respectively, and incubation was continued for another 24 h, after which the medium was changed and worms were incubated for an additional 24 h. Total RNA was extracted from untreated or TGF-β–treated worm pairs, and cDNA was synthesized and then subjected to PCR using different specific gene primers following the optimized PCR conditions for each gene employing the aforementioned protocol.

    Techniques: Transduction, In Vitro, Immunoprecipitation, Labeling, Incubation, Positive Control, Mutagenesis, SDS Page