recombinant human tgf β1  (R&D Systems)

 
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    Name:
    Recombinant Human TGF beta 1 Protein
    Description:
    The Recombinant Human TGF beta 1 Protein from R D Systems is derived from CHO The Recombinant Human TGF beta 1 Protein has been validated for the following applications Bioactivity
    Catalog Number:
    240-B-002
    Price:
    259
    Category:
    Proteins and Enzymes
    Source:
    CHO-derived Recombinant Human TGF-beta 1 Protein
    Applications:
    Bioactivity
    Purity:
    >97%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie« Blue Staining.
    Conjugate:
    Unconjugated
    Size:
    2 ug
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    Structured Review

    R&D Systems recombinant human tgf β1
    Recombinant Human TGF beta 1 Protein
    The Recombinant Human TGF beta 1 Protein from R D Systems is derived from CHO The Recombinant Human TGF beta 1 Protein 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 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human tgf β1 - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Roles of Stromal Cell RANKL, OPG, and M-CSF Expression in Biphasic TGF-β Regulation of Osteoclast Differentiation"

    Article Title: Roles of Stromal Cell RANKL, OPG, and M-CSF Expression in Biphasic TGF-β Regulation of Osteoclast Differentiation

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.20036

    OPG effects on differentiation stimulation. Osteoclasts were differentiated in co-cultures of bone marrow and ST2 cells with the addition of the indicated TGF-β1 concentration with or without 1 ng/ml OPG as indicated. TRAP positive multinucleated cells were counted and reported as treated/control. The values are an average of three replicate experiments with four wells per treatment in each experiment. * P
    Figure Legend Snippet: OPG effects on differentiation stimulation. Osteoclasts were differentiated in co-cultures of bone marrow and ST2 cells with the addition of the indicated TGF-β1 concentration with or without 1 ng/ml OPG as indicated. TRAP positive multinucleated cells were counted and reported as treated/control. The values are an average of three replicate experiments with four wells per treatment in each experiment. * P

    Techniques Used: Concentration Assay

    TGF-β1 regulation of macrophage colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) mRNAs. ST2 cells were untreated (NONE) or treated with 10 −5 M vitamin D and 10 −7 M Dexamethazone (vit D + Dex), with or without the indicated TGF-β1 concentration for 3 days and RNA was isolated and analyzed as described. The values are representative of two replicate experiments. * P
    Figure Legend Snippet: TGF-β1 regulation of macrophage colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) mRNAs. ST2 cells were untreated (NONE) or treated with 10 −5 M vitamin D and 10 −7 M Dexamethazone (vit D + Dex), with or without the indicated TGF-β1 concentration for 3 days and RNA was isolated and analyzed as described. The values are representative of two replicate experiments. * P

    Techniques Used: Concentration Assay, Isolation

    Transforming growth factor-beta 1 (TGF-β1) and TGF-β2 influences on osteoclast differentiation in the presence of stromal support cells. Differentiation of osteoclasts from marrow ( A , C , E ) and spleen ( B , D ) were assessed as detailed in the absence (control) or presence of the indicated TGF-β1 (A, B, E) or TGF-β2 (C, D) concentration. Tartrate resistant acid phosphatase (TRAP) positive multinucleated cells were counted and reported as treated/control. The values are an average of 3–6 replicate experiments with at least four wells per dose in each experiment. * P
    Figure Legend Snippet: Transforming growth factor-beta 1 (TGF-β1) and TGF-β2 influences on osteoclast differentiation in the presence of stromal support cells. Differentiation of osteoclasts from marrow ( A , C , E ) and spleen ( B , D ) were assessed as detailed in the absence (control) or presence of the indicated TGF-β1 (A, B, E) or TGF-β2 (C, D) concentration. Tartrate resistant acid phosphatase (TRAP) positive multinucleated cells were counted and reported as treated/control. The values are an average of 3–6 replicate experiments with at least four wells per dose in each experiment. * P

    Techniques Used: Concentration Assay

    M-CSF and RANKL effects on differentiation repression. Osteoclasts were differentiated in co-cultures of bone marrow and ST2 cells in the absence or presence of 2 ng/ml TGF-β1 with or without 25 ng/ml M-CSF and/or 60 ng/ml RANKL as indicated. TRAP positive multinucleated cells were counted and reported as treated/control. The values are an average of two replicate experiments with three wells per treatment in each experiment. * P
    Figure Legend Snippet: M-CSF and RANKL effects on differentiation repression. Osteoclasts were differentiated in co-cultures of bone marrow and ST2 cells in the absence or presence of 2 ng/ml TGF-β1 with or without 25 ng/ml M-CSF and/or 60 ng/ml RANKL as indicated. TRAP positive multinucleated cells were counted and reported as treated/control. The values are an average of two replicate experiments with three wells per treatment in each experiment. * P

    Techniques Used:

    Differentiation in the absence of stromal support cells. Precursors from marrow and spleen were cultured with M-CSF (25 ng/ml) and RANKL (30 ng/ml) in the presence or absence of TGF-β1 as described for 9 days and TRAP stained. Differentiation of cells from spleen and marrow were assessed as detailed. TRAP positive multinucleated cells were counted as outlined. The values are an average of three replicate experiments with at least four wells per dose in each experiment. * P
    Figure Legend Snippet: Differentiation in the absence of stromal support cells. Precursors from marrow and spleen were cultured with M-CSF (25 ng/ml) and RANKL (30 ng/ml) in the presence or absence of TGF-β1 as described for 9 days and TRAP stained. Differentiation of cells from spleen and marrow were assessed as detailed. TRAP positive multinucleated cells were counted as outlined. The values are an average of three replicate experiments with at least four wells per dose in each experiment. * P

    Techniques Used: Cell Culture, Staining

    2) 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

    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 "TGF-β1/Smad Signaling Pathway Regulates Epithelial-to-Mesenchymal Transition in Esophageal Squamous Cell Carcinoma: In Vitro and Clinical Analyses of Cell Lines and Nomadic Kazakh Patients from Northwest Xinjiang, China"

    Article Title: TGF-β1/Smad Signaling Pathway Regulates Epithelial-to-Mesenchymal Transition in Esophageal Squamous Cell Carcinoma: In Vitro and Clinical Analyses of Cell Lines and Nomadic Kazakh Patients from Northwest Xinjiang, China

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112300

    Representative immunohistochemical staining for TGF-β1 (A1,A2), TGF-β RII (B1,B2), p-Smad2/3 (C1,C2), E-cadherin(D1,D2), N-cadherin(E1,E2), and vimentin(F1,F2) in ESCC and NCAT tissues (200×). Scale bars represent 100 µm.
    Figure Legend Snippet: Representative immunohistochemical staining for TGF-β1 (A1,A2), TGF-β RII (B1,B2), p-Smad2/3 (C1,C2), E-cadherin(D1,D2), N-cadherin(E1,E2), and vimentin(F1,F2) in ESCC and NCAT tissues (200×). Scale bars represent 100 µm.

    Techniques Used: Immunohistochemistry, Staining

    Morphological changes of Eca109(1–1) and KYSE150(1–2) cells after TGF-β1 treatment. A: Untreated Eca109 and KYSE150 cells, 100×; B,C: Eca109 and KYSE150 cells treated with TGF-β1 (1,5 ng/mL) for 36 hrs, 100×. D Control stimulation with TGF-β1 (5 ng/mL) in the presence of SB4315425(5 µM) for 36 hrs, 100×. Scale bars represent 50 µm.
    Figure Legend Snippet: Morphological changes of Eca109(1–1) and KYSE150(1–2) cells after TGF-β1 treatment. A: Untreated Eca109 and KYSE150 cells, 100×; B,C: Eca109 and KYSE150 cells treated with TGF-β1 (1,5 ng/mL) for 36 hrs, 100×. D Control stimulation with TGF-β1 (5 ng/mL) in the presence of SB4315425(5 µM) for 36 hrs, 100×. Scale bars represent 50 µm.

    Techniques Used:

    Western blot analysis of E-cadherin, N-cadherin, vimentin, P-Smad2 protein expression in Eca109(2A–D), Eca9706(2E–H), KYSE150(2I–L) cells treated with TGF-β1 or SB431542 at differential concentrations. A . Effects of treatment of Eca109 cells with TGF-β1 (1, 5, or 10 ng/mL) on the expression of E-cadherin (molecular weight 97 kDa), N-cadherin (molecular weight 100 kDa), vimentin (molecular weight 57 kDa), p-Smad2 (molecular weight 52 kDa) and Smad7 (molecular weight 51 kDa) by Western Blot. B . Quantiative analysis of treatment of Eca109 cells with TGF-β1 (1, 5, or 10 ng/mL), E-cadherin, N-cadherin, vimentin, P-Smad2 and Smad7 expression levels; Y axis: banding densities of test marker versus β-actin. Data are expressed as a significant change relative to the control. Each bar represents the mean±s.d. *, p
    Figure Legend Snippet: Western blot analysis of E-cadherin, N-cadherin, vimentin, P-Smad2 protein expression in Eca109(2A–D), Eca9706(2E–H), KYSE150(2I–L) cells treated with TGF-β1 or SB431542 at differential concentrations. A . Effects of treatment of Eca109 cells with TGF-β1 (1, 5, or 10 ng/mL) on the expression of E-cadherin (molecular weight 97 kDa), N-cadherin (molecular weight 100 kDa), vimentin (molecular weight 57 kDa), p-Smad2 (molecular weight 52 kDa) and Smad7 (molecular weight 51 kDa) by Western Blot. B . Quantiative analysis of treatment of Eca109 cells with TGF-β1 (1, 5, or 10 ng/mL), E-cadherin, N-cadherin, vimentin, P-Smad2 and Smad7 expression levels; Y axis: banding densities of test marker versus β-actin. Data are expressed as a significant change relative to the control. Each bar represents the mean±s.d. *, p

    Techniques Used: Western Blot, Expressing, Molecular Weight, Marker

    Transwell assay of Eca109 cells with or without TGF-β1 treatment. A: Untreated Eca109 cells, 200×. B: Eca109 cells treated with TGF-β1 (10 ng/mL) for 24 h, 200×. C: The number of invading cells: Untreated Eca109 cells vs. Eca109 cells treated with TGFβ1. Data shown represent the average of 3 independent experiments. * p
    Figure Legend Snippet: Transwell assay of Eca109 cells with or without TGF-β1 treatment. A: Untreated Eca109 cells, 200×. B: Eca109 cells treated with TGF-β1 (10 ng/mL) for 24 h, 200×. C: The number of invading cells: Untreated Eca109 cells vs. Eca109 cells treated with TGFβ1. Data shown represent the average of 3 independent experiments. * p

    Techniques Used: Transwell Assay

    5) Product Images from "Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis *"

    Article Title: Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.691519

    Structural and mechanical integrity of formed collagen fibril networks in Fn-null hepatic stellate cells. A , elastic modulus of collagen fibrils in Fn-null hepatic stellate cells without treatment ( Ntx ) or with TGF-β1 or TGF-β1 plus 200
    Figure Legend Snippet: Structural and mechanical integrity of formed collagen fibril networks in Fn-null hepatic stellate cells. A , elastic modulus of collagen fibrils in Fn-null hepatic stellate cells without treatment ( Ntx ) or with TGF-β1 or TGF-β1 plus 200

    Techniques Used:

    6) Product Images from "Targeting of dermal myofibroblasts through death receptor 5 arrests fibrosis in mouse models of scleroderma"

    Article Title: Targeting of dermal myofibroblasts through death receptor 5 arrests fibrosis in mouse models of scleroderma

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09101-4

    TGF-β signaling regulates the DR5 expression in HDFs though Smad2/3-SP1 complexes. a Measurements of DR5 promoter activity with the dual-luciferase-reporter system in HDFs treated with SB203580 (10 μM, Smad2 inhibitor) and SIS3 (5 μM, Smad3 inhibitor) for 2 h followed by TGF-β1 (10 ng/mL) treatment for 54 h ( n = 6 biologically independent experiments). b , c HDFs were transfected with SP1 and/or Smad 2/3 siRNA for 24 h. The cells were then exposed to TGF-β1 for 54 h. b mRNA DR5 levels in siRNA transfected HDFs with and without TGF-β1 treatment ( n = 4 biologically independent experiments). c Effects of siRNA-mediated knockdowns of SP1 and Smad2/3 on TGF-β1-induced DR5 levels in HDFs by western blot. Relative DR5 protein levels normalized to GAPDH ( n = 4 biologically independent experiments). d Western blot analysis of Smad2/3 immunoprecipitates (IP) of HDFs with or without TGF-β1 treatment ( n = 3 biologically independent experiments). e Binding assessment with ChIP assays of the two potential SP1 sites (site 1: −195 to −190, site 2: −159 to −154) and non-target site (NT) to the DR5 promoter in HDFs treated with TGF-β1 ( n = 4). f A representation of TGF-β pathway including Smad2/3, Smad4, and SP1 complexes in the translation mechanism of DR5. Data are shown as median ± interquartile range. The Mann–Whitney test was used. * P
    Figure Legend Snippet: TGF-β signaling regulates the DR5 expression in HDFs though Smad2/3-SP1 complexes. a Measurements of DR5 promoter activity with the dual-luciferase-reporter system in HDFs treated with SB203580 (10 μM, Smad2 inhibitor) and SIS3 (5 μM, Smad3 inhibitor) for 2 h followed by TGF-β1 (10 ng/mL) treatment for 54 h ( n = 6 biologically independent experiments). b , c HDFs were transfected with SP1 and/or Smad 2/3 siRNA for 24 h. The cells were then exposed to TGF-β1 for 54 h. b mRNA DR5 levels in siRNA transfected HDFs with and without TGF-β1 treatment ( n = 4 biologically independent experiments). c Effects of siRNA-mediated knockdowns of SP1 and Smad2/3 on TGF-β1-induced DR5 levels in HDFs by western blot. Relative DR5 protein levels normalized to GAPDH ( n = 4 biologically independent experiments). d Western blot analysis of Smad2/3 immunoprecipitates (IP) of HDFs with or without TGF-β1 treatment ( n = 3 biologically independent experiments). e Binding assessment with ChIP assays of the two potential SP1 sites (site 1: −195 to −190, site 2: −159 to −154) and non-target site (NT) to the DR5 promoter in HDFs treated with TGF-β1 ( n = 4). f A representation of TGF-β pathway including Smad2/3, Smad4, and SP1 complexes in the translation mechanism of DR5. Data are shown as median ± interquartile range. The Mann–Whitney test was used. * P

    Techniques Used: Expressing, Activity Assay, Luciferase, Transfection, Western Blot, Binding Assay, Chromatin Immunoprecipitation, MANN-WHITNEY

    Mouse anti-DR5 antibody (MD5-1) reverses skin fibrosis in bleomycin (BLM)-induced mouse model of skin fibrosis. DBA2/J mice were divided into three groups: the control group treated with NaCl (s.c., day 0–42) and IgG (5 mg/kg, i.p., every other day, day 22–42, n = 8) and the BLM-induced groups treated with BLM (s.c., day 0–21) and IgG (5 mg/kg, i.p., every other day, day 22–42, n = 9) or MD5-1 (5 mg/kg, i.p., every other day, day 22–42, n = 8). a Representative images of H E and Trichrome-stained skin sections ( n = 8 mice treated with NaCl/IgG, n = 9 BLM/IgG, n = 8 BLM/MD5-1, biologically independent animals; scale bars, 100 µm). b Analysis of dermal thickness and hydroxyproline content. c Representative immunostaining for α-SMA (green) and nuclei (DAPI, blue) ( n = 8 biologically independent animals; scale bars, 50 µm). d Myofibroblast counts in the skins of control and BLM-induced groups ( n = 8 biologically independent animals). e Measurement of caspase-8 and caspase-3/7 activities in the skins of control and BLM-induced groups ( n = 7 biologically independent animals). f qPCR analysis of mRNA ACTA2 , Col1a1 , Col1a2 , TGF-β1 , PDGFR-β, and PDGFα in the skin ( n = 8 biologically independent animals). Data are shown as median ± interquartile range. The Mann–Whitney test was used. * P
    Figure Legend Snippet: Mouse anti-DR5 antibody (MD5-1) reverses skin fibrosis in bleomycin (BLM)-induced mouse model of skin fibrosis. DBA2/J mice were divided into three groups: the control group treated with NaCl (s.c., day 0–42) and IgG (5 mg/kg, i.p., every other day, day 22–42, n = 8) and the BLM-induced groups treated with BLM (s.c., day 0–21) and IgG (5 mg/kg, i.p., every other day, day 22–42, n = 9) or MD5-1 (5 mg/kg, i.p., every other day, day 22–42, n = 8). a Representative images of H E and Trichrome-stained skin sections ( n = 8 mice treated with NaCl/IgG, n = 9 BLM/IgG, n = 8 BLM/MD5-1, biologically independent animals; scale bars, 100 µm). b Analysis of dermal thickness and hydroxyproline content. c Representative immunostaining for α-SMA (green) and nuclei (DAPI, blue) ( n = 8 biologically independent animals; scale bars, 50 µm). d Myofibroblast counts in the skins of control and BLM-induced groups ( n = 8 biologically independent animals). e Measurement of caspase-8 and caspase-3/7 activities in the skins of control and BLM-induced groups ( n = 7 biologically independent animals). f qPCR analysis of mRNA ACTA2 , Col1a1 , Col1a2 , TGF-β1 , PDGFR-β, and PDGFα in the skin ( n = 8 biologically independent animals). Data are shown as median ± interquartile range. The Mann–Whitney test was used. * P

    Techniques Used: Mouse Assay, Staining, Immunostaining, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    Myofibroblasts (MFBs) differentiated from primary human dermal fibroblasts (HDFs) become sensitive to death receptor (DR)-mediated apoptosis through upregulated DR5. a RNA-seq data of skin biopsies of patients with systemic sclerosis (SSc) demonstrated upregulated mRNA ACTA2 , DR4 , DR5 , TRAIL , and other fibrogenic components compared to normal skin ( n = 4 biologically independent samples). b Relative mRNA DR4 and DR5 expression from the skin of patients with SSc and morphea ( n = 5 biologically independent samples). c Representative double-immunostaining for DR4 (top, green), DR5 (bottom, green), α-SMA (red), and nuclei (DAPI, blue) in the healthy and fibrotic skin samples (scale bars, 10 µm). The fluorescence intensity was measured by ImageJ software ( n = 5 biologically independent samples). d Relative mRNA DR4 and DR5 expression from normal and fibrotic fibroblasts isolated from patients ( n = 7 normal fibroblast, n = 9 fibrotic fibroblasts biologically independent samples). e Relative mRNA DR4 and DR5 expression in HDFs treated with TGF-β1 (10 ng/mL) ( n = 6 biologically independent experiments). f Western blot analysis of DR4, DR5, α-SMA, and PDGFR-β in HDFs treated with TGF-β1 (10 ng/mL). Quantification of protein levels normalized to GAPDH ( n = 3 biologically independent experiments). g Representative immunoblots of apoptosis markers cleaved (Cl.) caspase-8, caspase-3, and PARP-1 in TGF-β1 (10 ng/mL for 54 h) activated HDFs treated with TLY012 (1 μg/mL) for 6 h ( n = 3 biologically independent experiments). h Caspase-8 and 3/7 activity in TGF-β1 (10 ng/mL for 54 h) activated HDFs treated with TLY012 (1 μg/mL) for 6 h ( n = 4 biologically independent experiments). i Knockdown effects of DR4 and DR5 on TLY012-induced apoptosis in HDFs activated by TGF-β1 (10 ng/mL for 54 h) as shown by western blot analysis ( n = 4 biologically independent experiments). j Flow cytometry analysis to determine the surface expression of DR4 and DR5 in HDFs activated by TGF-β1 for 54 h ( n = 3 biologically independent experiments). Data are shown as median ± interquartile range. The Mann–Whitney test was used. * P
    Figure Legend Snippet: Myofibroblasts (MFBs) differentiated from primary human dermal fibroblasts (HDFs) become sensitive to death receptor (DR)-mediated apoptosis through upregulated DR5. a RNA-seq data of skin biopsies of patients with systemic sclerosis (SSc) demonstrated upregulated mRNA ACTA2 , DR4 , DR5 , TRAIL , and other fibrogenic components compared to normal skin ( n = 4 biologically independent samples). b Relative mRNA DR4 and DR5 expression from the skin of patients with SSc and morphea ( n = 5 biologically independent samples). c Representative double-immunostaining for DR4 (top, green), DR5 (bottom, green), α-SMA (red), and nuclei (DAPI, blue) in the healthy and fibrotic skin samples (scale bars, 10 µm). The fluorescence intensity was measured by ImageJ software ( n = 5 biologically independent samples). d Relative mRNA DR4 and DR5 expression from normal and fibrotic fibroblasts isolated from patients ( n = 7 normal fibroblast, n = 9 fibrotic fibroblasts biologically independent samples). e Relative mRNA DR4 and DR5 expression in HDFs treated with TGF-β1 (10 ng/mL) ( n = 6 biologically independent experiments). f Western blot analysis of DR4, DR5, α-SMA, and PDGFR-β in HDFs treated with TGF-β1 (10 ng/mL). Quantification of protein levels normalized to GAPDH ( n = 3 biologically independent experiments). g Representative immunoblots of apoptosis markers cleaved (Cl.) caspase-8, caspase-3, and PARP-1 in TGF-β1 (10 ng/mL for 54 h) activated HDFs treated with TLY012 (1 μg/mL) for 6 h ( n = 3 biologically independent experiments). h Caspase-8 and 3/7 activity in TGF-β1 (10 ng/mL for 54 h) activated HDFs treated with TLY012 (1 μg/mL) for 6 h ( n = 4 biologically independent experiments). i Knockdown effects of DR4 and DR5 on TLY012-induced apoptosis in HDFs activated by TGF-β1 (10 ng/mL for 54 h) as shown by western blot analysis ( n = 4 biologically independent experiments). j Flow cytometry analysis to determine the surface expression of DR4 and DR5 in HDFs activated by TGF-β1 for 54 h ( n = 3 biologically independent experiments). Data are shown as median ± interquartile range. The Mann–Whitney test was used. * P

    Techniques Used: RNA Sequencing Assay, Expressing, Double Immunostaining, Fluorescence, Software, Isolation, Western Blot, Activity Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    7) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus-Encoded MicroRNA miR-K12-11 Attenuates Transforming Growth Factor Beta Signaling through Suppression of SMAD5"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus-Encoded MicroRNA miR-K12-11 Attenuates Transforming Growth Factor Beta Signaling through Suppression of SMAD5

    Journal: Journal of Virology

    doi: 10.1128/JVI.06245-11

    SMAD5 was the predominant target of miR-K12-11. (A) miR-K12-11 downregulates SMAD1/2/3/5 3′ UTR reporter activity in HEK293T cells. One hundred nanograms pGL3-SMAD1- 3′ UTR, pGL3-SMAD2- 3′ UTR, pGL3-SMAD3- 3′ UTR, or pGL3-SMAD5- 3′ UTR was cotransfected with either pCDH-miR-K12-11 or pCDH-copGFP (1 μg) into HEK293T cells. The pGL3-BACH1 3′ UTR was used as a positive control. (B) Western blotting of SMAD1/2/3/5 in Ramos-teton-miR-K12-11 cells after induction with or without doxycycline for the indicated times. As a loading control, β-actin was also detected in the same blotting. Values represent percentages of SMAD1/2/3/5 normalized against β-actin and compared with an untreated control. (C) Total RNA of the same samples was reverse-transcribed and then used as a template to determine the SMAD5 mRNA levels by standard qRT-PCR. Values were normalized against β-actin. (D) Ramos-teton-miR-K12-11 cells were cultured with or without doxycycline for 48 h and then starved for 12 h before the addition of TGF-β1 (10 ng/ml). Lysates of these cells were taken 1 or 2 h after TGF-β1 stimulation to quantify the levels of phospho-SMAD1/5 and SMAD5 proteins by Western blotting. As a normalizing control, β-actin was also detected in the same blotting. Densitometry values represent the percentage of p-SMAD1/5 normalized against β-actin and compared with an uninduced control.
    Figure Legend Snippet: SMAD5 was the predominant target of miR-K12-11. (A) miR-K12-11 downregulates SMAD1/2/3/5 3′ UTR reporter activity in HEK293T cells. One hundred nanograms pGL3-SMAD1- 3′ UTR, pGL3-SMAD2- 3′ UTR, pGL3-SMAD3- 3′ UTR, or pGL3-SMAD5- 3′ UTR was cotransfected with either pCDH-miR-K12-11 or pCDH-copGFP (1 μg) into HEK293T cells. The pGL3-BACH1 3′ UTR was used as a positive control. (B) Western blotting of SMAD1/2/3/5 in Ramos-teton-miR-K12-11 cells after induction with or without doxycycline for the indicated times. As a loading control, β-actin was also detected in the same blotting. Values represent percentages of SMAD1/2/3/5 normalized against β-actin and compared with an untreated control. (C) Total RNA of the same samples was reverse-transcribed and then used as a template to determine the SMAD5 mRNA levels by standard qRT-PCR. Values were normalized against β-actin. (D) Ramos-teton-miR-K12-11 cells were cultured with or without doxycycline for 48 h and then starved for 12 h before the addition of TGF-β1 (10 ng/ml). Lysates of these cells were taken 1 or 2 h after TGF-β1 stimulation to quantify the levels of phospho-SMAD1/5 and SMAD5 proteins by Western blotting. As a normalizing control, β-actin was also detected in the same blotting. Densitometry values represent the percentage of p-SMAD1/5 normalized against β-actin and compared with an uninduced control.

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

    Ectopic expression of miR-K12-11 attenuated the cytostatic effect of TGF-β1 in Ramos cells. (A) Ramos-teton-miR-K12-11 cells were treated with doxycycline to upregulate the expression of miR-K12-11. RNA from these samples was reverse-transcribed and then used to quantify the mature miR-K12-11 and housekeeping U6 levels with a bulge-loop qRT-PCR. KSHV-positive cell lines, BC3 or JSC, were used as a positive control. All values are expressed as fold induction of the untreated Ramos cells. (B) Growth of Ramos cells overexpressing miR-K12-11 or control miRNA treated with either TGF-β1 or vehicle over 3 days. Error bars represent SDs. (C) Cell cycle analyses show G 0 /G 1 arrest after TGF-β1 exposure in Ramos cells overexpressing control miRNA, miR-K12-11, or miR-K12-11 in combination with SMAD5. The corresponding P value was calculated for each group, and P values are shown at the top (Student's t test).
    Figure Legend Snippet: Ectopic expression of miR-K12-11 attenuated the cytostatic effect of TGF-β1 in Ramos cells. (A) Ramos-teton-miR-K12-11 cells were treated with doxycycline to upregulate the expression of miR-K12-11. RNA from these samples was reverse-transcribed and then used to quantify the mature miR-K12-11 and housekeeping U6 levels with a bulge-loop qRT-PCR. KSHV-positive cell lines, BC3 or JSC, were used as a positive control. All values are expressed as fold induction of the untreated Ramos cells. (B) Growth of Ramos cells overexpressing miR-K12-11 or control miRNA treated with either TGF-β1 or vehicle over 3 days. Error bars represent SDs. (C) Cell cycle analyses show G 0 /G 1 arrest after TGF-β1 exposure in Ramos cells overexpressing control miRNA, miR-K12-11, or miR-K12-11 in combination with SMAD5. The corresponding P value was calculated for each group, and P values are shown at the top (Student's t test).

    Techniques Used: Expressing, Quantitative RT-PCR, Positive Control

    8) Product Images from "The RUNX3 Tumor Suppressor Upregulates Bim in Gastric Epithelial Cells Undergoing Transforming Growth Factor ?-Induced Apoptosis"

    Article Title: The RUNX3 Tumor Suppressor Upregulates Bim in Gastric Epithelial Cells Undergoing Transforming Growth Factor ?-Induced Apoptosis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01926-05

    Expression of Runx3, TGF-β1, and TGF-β RI/II in gastric epithelial cells of neonate mice and lack of Bim expression in TGF-β1 −/− embryonic day 18.5 stomach. (A) Expression of Runx3 in WT and Runx3 −/− stomachs revealed by immunostaining with R3-1E10. Immunostaining with normal mouse IgG as a primary antibody in a WT stomach is shown as a negative control. (B) Expression of TGF-β1 , TGFβRI , and TGFβRII in WT and Runx3 −/− stomachs as revealed by Northern blotting. (C) Expression of TGF-β1 and TGF-β RI/II in WT and Runx3 −/− stomachs. Immunostaining with normal mouse IgG as a primary antibody is shown as a negative control (control). (D) Expression of Bim in WT and TGF-β1 −/− stomachs. Immunostaining with normal mouse IgG as a primary antibody in a WT stomach is shown as a negative control (IgG). Counter-staining was done with hematoxylin. Scale bar, 200 μm.
    Figure Legend Snippet: Expression of Runx3, TGF-β1, and TGF-β RI/II in gastric epithelial cells of neonate mice and lack of Bim expression in TGF-β1 −/− embryonic day 18.5 stomach. (A) Expression of Runx3 in WT and Runx3 −/− stomachs revealed by immunostaining with R3-1E10. Immunostaining with normal mouse IgG as a primary antibody in a WT stomach is shown as a negative control. (B) Expression of TGF-β1 , TGFβRI , and TGFβRII in WT and Runx3 −/− stomachs as revealed by Northern blotting. (C) Expression of TGF-β1 and TGF-β RI/II in WT and Runx3 −/− stomachs. Immunostaining with normal mouse IgG as a primary antibody is shown as a negative control (control). (D) Expression of Bim in WT and TGF-β1 −/− stomachs. Immunostaining with normal mouse IgG as a primary antibody in a WT stomach is shown as a negative control (IgG). Counter-staining was done with hematoxylin. Scale bar, 200 μm.

    Techniques Used: Expressing, Mouse Assay, Immunostaining, Negative Control, Northern Blot, Staining

    9) Product Images from "TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade"

    Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0189-8

    Regulation of Trx and GSTM1 promoter activity by TGF-β downregulation via a Smad complex. a A375 and HPAC cell lines were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, Trx and GSTM1 promoter activities were analyzed by Chip assays using antibodies to AP-1 or Sp1. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p
    Figure Legend Snippet: Regulation of Trx and GSTM1 promoter activity by TGF-β downregulation via a Smad complex. a A375 and HPAC cell lines were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, Trx and GSTM1 promoter activities were analyzed by Chip assays using antibodies to AP-1 or Sp1. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p

    Techniques Used: Activity Assay, Infection, Expressing, Chromatin Immunoprecipitation

    Involvement of reciprocal ER stress and SAPK activation during TGF-β downregulation-induced cell death. a A375 cells were infected with adenovirus expressing shTGF-β1 or -β2. After 48 h, various ER stress-related proteins were detected by western blot analysis. b A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then cell viability was tested using an MTS viability assay (left). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (** p
    Figure Legend Snippet: Involvement of reciprocal ER stress and SAPK activation during TGF-β downregulation-induced cell death. a A375 cells were infected with adenovirus expressing shTGF-β1 or -β2. After 48 h, various ER stress-related proteins were detected by western blot analysis. b A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then cell viability was tested using an MTS viability assay (left). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (** p

    Techniques Used: Activation Assay, Infection, Expressing, Western Blot, Viability Assay

    Antitumor effect and schematic diagram of cancer cell death by TGF-β downregulation. a BALB/c athymic nude mice were injected with 8 × 10 6 A375 or HPAC cells in 100 μL. When the tumors reached an average size of 60–80 mm 3 , the nude mice received intratumoral injections of 1 × 10 9 plaque-forming units (pfu) of various kinds of adenovirus (Ad-NC, Ad-shTGF-β1, or Ad-shTGF-β2) in 50 μL of PBS or PBS alone on days 1, 3, and 5. Tumor volume was monitored and recorded every 2 days until the end of the study. Values represent the mean ± SE (five animals per group) (top). Asterisks indicate a significant difference compared to each given control (* p
    Figure Legend Snippet: Antitumor effect and schematic diagram of cancer cell death by TGF-β downregulation. a BALB/c athymic nude mice were injected with 8 × 10 6 A375 or HPAC cells in 100 μL. When the tumors reached an average size of 60–80 mm 3 , the nude mice received intratumoral injections of 1 × 10 9 plaque-forming units (pfu) of various kinds of adenovirus (Ad-NC, Ad-shTGF-β1, or Ad-shTGF-β2) in 50 μL of PBS or PBS alone on days 1, 3, and 5. Tumor volume was monitored and recorded every 2 days until the end of the study. Values represent the mean ± SE (five animals per group) (top). Asterisks indicate a significant difference compared to each given control (* p

    Techniques Used: Mouse Assay, Injection

    TGF-β downregulation and ROS generation by TGF-β shRNAs. A Downregulation of TGF-β1 and TGF-β2 after infection with adenoviruses expressing TGF-β shRNAs. a Schematic structure of adenoviral vectors expressing TGF-β shRNA. dl324-∆E1A-∆E1B-∆E3-IX-U6-NC (Ad-NC) is a replication-incompetent adenovirus used as the negative control. It contains the scrambled DNA sequence for shRNA, which is under the control of the U6 promoter. dl324-∆E1A-∆E1B∆E3-IX-U6-shTGF-β1 or -β2 (Ad-shTGF-β1 or -β2) is a replication-incompetent adenovirus expressing human TGF-β1 or -β2 shRNA. NC negative control. b Downregulation of human transforming growth factor β1 (TGF-β1) or β2 by adenovirus expressing shTGF-β1 or -β2. Human A375 and HPAC cells were infected with various MOIs of adenovirus expressing shRNA targeting human TGF-β1 (Ad-shTGF-β1) or human TGF-β2 (Ad-shTGF-β2) or scrambled RNA (Ad-NC). TGF-β1 or -β2 mRNA was assayed by quantitative real-time polymerase chain reaction (qRT-PCR), and c protein levels were assayed by enzyme-linked immunosorbent assay (ELISA). MOI multiplicity of infection, NC negative control. Error bars represent the standard error from three independent experiments. B Various cancer cell lines were treated with adenovirus expressing shTGF-β1 or -β2 for 48 h and incubated for an additional 14 days for clonogenic assays. The numbers indicate the relative ratio of clone numbers to those of Ad-NC. C Global transcriptome analysis of TGF-β downregulation. Expression values of differentially expressed genes are shown in heat map format. Gene expression levels are visualized as row standardized z scores ranging from green (−1) to red (+1) across all samples. The rows are organized by hierarchical clustering analysis with complete linkage and Euclidean distance as a measure of similarity from samples of A375 cells (NC, shTGF-β1, shTGF-β2) (Left) and HPAC cells (NC, shTGF-β1, shTGF-β2) (Right). NC, A375, and HPAC cells infected with adenovirus expressing nonsense shRNA as a negative control at 100 MOI; shTGF-β1 or -β2; A375 and HPAC cells infected with adenoviruexpressing TGF-β1 or -β2 shRNA at 100 MOI. D Effect of adenovirus expressing shTGF-β1 or shTGF-β2 in various cancer cell lines. Each cell line was treated with adenovirus expressing shTGF-β1 or shTGF-β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. E Pancreatic normal cell lines were treated with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. F ROS generation was induced by shTGF-β1- and shTGF-β2-expressing adenoviruses. A375 cells, HPAC cancer cells, and pancreatic normal cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively, after 48 h of incubation with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. Cell viability was tested via an MTS viability assay after 48 h of infection. Error bars represent the standard error from three independent experiments. p Values
    Figure Legend Snippet: TGF-β downregulation and ROS generation by TGF-β shRNAs. A Downregulation of TGF-β1 and TGF-β2 after infection with adenoviruses expressing TGF-β shRNAs. a Schematic structure of adenoviral vectors expressing TGF-β shRNA. dl324-∆E1A-∆E1B-∆E3-IX-U6-NC (Ad-NC) is a replication-incompetent adenovirus used as the negative control. It contains the scrambled DNA sequence for shRNA, which is under the control of the U6 promoter. dl324-∆E1A-∆E1B∆E3-IX-U6-shTGF-β1 or -β2 (Ad-shTGF-β1 or -β2) is a replication-incompetent adenovirus expressing human TGF-β1 or -β2 shRNA. NC negative control. b Downregulation of human transforming growth factor β1 (TGF-β1) or β2 by adenovirus expressing shTGF-β1 or -β2. Human A375 and HPAC cells were infected with various MOIs of adenovirus expressing shRNA targeting human TGF-β1 (Ad-shTGF-β1) or human TGF-β2 (Ad-shTGF-β2) or scrambled RNA (Ad-NC). TGF-β1 or -β2 mRNA was assayed by quantitative real-time polymerase chain reaction (qRT-PCR), and c protein levels were assayed by enzyme-linked immunosorbent assay (ELISA). MOI multiplicity of infection, NC negative control. Error bars represent the standard error from three independent experiments. B Various cancer cell lines were treated with adenovirus expressing shTGF-β1 or -β2 for 48 h and incubated for an additional 14 days for clonogenic assays. The numbers indicate the relative ratio of clone numbers to those of Ad-NC. C Global transcriptome analysis of TGF-β downregulation. Expression values of differentially expressed genes are shown in heat map format. Gene expression levels are visualized as row standardized z scores ranging from green (−1) to red (+1) across all samples. The rows are organized by hierarchical clustering analysis with complete linkage and Euclidean distance as a measure of similarity from samples of A375 cells (NC, shTGF-β1, shTGF-β2) (Left) and HPAC cells (NC, shTGF-β1, shTGF-β2) (Right). NC, A375, and HPAC cells infected with adenovirus expressing nonsense shRNA as a negative control at 100 MOI; shTGF-β1 or -β2; A375 and HPAC cells infected with adenoviruexpressing TGF-β1 or -β2 shRNA at 100 MOI. D Effect of adenovirus expressing shTGF-β1 or shTGF-β2 in various cancer cell lines. Each cell line was treated with adenovirus expressing shTGF-β1 or shTGF-β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. E Pancreatic normal cell lines were treated with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. F ROS generation was induced by shTGF-β1- and shTGF-β2-expressing adenoviruses. A375 cells, HPAC cancer cells, and pancreatic normal cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively, after 48 h of incubation with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. Cell viability was tested via an MTS viability assay after 48 h of infection. Error bars represent the standard error from three independent experiments. p Values

    Techniques Used: Infection, Expressing, shRNA, Negative Control, Sequencing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Microscopy, Viability Assay

    NOX4 is responsible for TGF-β downregulation-induced ROS generation. a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of NOX1, NOX4, 5-lipoxygenase, and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or transfected with pCMV6-myr-Akt or infected with adenovirus expressing shTGF-β1 and subsequently transfected with pCMV6-myr-Akt. After 48 h, NOX4, phospho-Akt, Akt, and GAPDH were detected by western blot analysis. c Expression levels of NOX4 mRNA in A375 and HPAC cells were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). d A375 cells were infected with adenovirus expressing shTGF-β1. After 6 h, infected cells were treated with NAC (10 mM) for 42 h, and then the expression of NOX4 and GAPDH was detected by western blot analysis. e A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with p38 inhibitor (SB203580, 10 μM) or JNK inhibitor (SP600125, 10 μM) or both for 42 h. Then the expression of NOX4 and GAPDH was detected by western blot analysis. f A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by NAC (10 mM) or adenovirus expressing shTGF-β1 followed by p38 inhibitor (SB203580, 10 μM) or adenovirus expressing shTGF-β1 followed by transfection with pCMV6-myr-Akt or adenovirus expressing shTGF-β1 followed by transfection with siRNA of Smad4 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, the expression levels of NOX4 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p
    Figure Legend Snippet: NOX4 is responsible for TGF-β downregulation-induced ROS generation. a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of NOX1, NOX4, 5-lipoxygenase, and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or transfected with pCMV6-myr-Akt or infected with adenovirus expressing shTGF-β1 and subsequently transfected with pCMV6-myr-Akt. After 48 h, NOX4, phospho-Akt, Akt, and GAPDH were detected by western blot analysis. c Expression levels of NOX4 mRNA in A375 and HPAC cells were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). d A375 cells were infected with adenovirus expressing shTGF-β1. After 6 h, infected cells were treated with NAC (10 mM) for 42 h, and then the expression of NOX4 and GAPDH was detected by western blot analysis. e A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with p38 inhibitor (SB203580, 10 μM) or JNK inhibitor (SP600125, 10 μM) or both for 42 h. Then the expression of NOX4 and GAPDH was detected by western blot analysis. f A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by NAC (10 mM) or adenovirus expressing shTGF-β1 followed by p38 inhibitor (SB203580, 10 μM) or adenovirus expressing shTGF-β1 followed by transfection with pCMV6-myr-Akt or adenovirus expressing shTGF-β1 followed by transfection with siRNA of Smad4 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, the expression levels of NOX4 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p

    Techniques Used: Infection, Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Decreases in Trx and GSTM1 expression and the formation of complexes with ASK1 after TGF-β downregulation. a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of p-ASK1, ASK1, Trx, GSTM1 and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of Trx and GSTM1 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p
    Figure Legend Snippet: Decreases in Trx and GSTM1 expression and the formation of complexes with ASK1 after TGF-β downregulation. a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of p-ASK1, ASK1, Trx, GSTM1 and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of Trx and GSTM1 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p

    Techniques Used: Expressing, Infection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    ASK1 functions as a key mediator of TGF-β downregulation-induced cell death triggered by non-canonical signaling Akt inactivation a ASK1 mediates TGF-β-induced cell death via p38/JNK activation. A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 (100 MOI) and subsequently transfected with siASK1. siASK1 transfection was based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested by an MTS viability assay. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p
    Figure Legend Snippet: ASK1 functions as a key mediator of TGF-β downregulation-induced cell death triggered by non-canonical signaling Akt inactivation a ASK1 mediates TGF-β-induced cell death via p38/JNK activation. A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 (100 MOI) and subsequently transfected with siASK1. siASK1 transfection was based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested by an MTS viability assay. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p

    Techniques Used: Activation Assay, Infection, Expressing, Transfection, Viability Assay

    10) Product Images from "Lactoferrin Combined with Retinoic Acid Stimulates B1 Cells to Express IgA Isotype and Gut-homing Molecules"

    Article Title: Lactoferrin Combined with Retinoic Acid Stimulates B1 Cells to Express IgA Isotype and Gut-homing Molecules

    Journal: Immune Network

    doi: 10.4110/in.2015.15.1.37

    Effect of LF, RA, and TGF-β1 on Ig secretion by mouse peritoneal B1 and B2 cells. Mouse peritoneal B1 and B2 cells were stimulated with LPS (12.5 µg/ml), RA (25 nM), LF (60 µg/ml), and TGF-β1 (0.2 ng/ml) for 7 days. Supernatants were collected, and Ig production was determined by isotype-specific ELISA. Data represent the results of one of the two independent experiments and are means of triplicated samples±SEM. * p
    Figure Legend Snippet: Effect of LF, RA, and TGF-β1 on Ig secretion by mouse peritoneal B1 and B2 cells. Mouse peritoneal B1 and B2 cells were stimulated with LPS (12.5 µg/ml), RA (25 nM), LF (60 µg/ml), and TGF-β1 (0.2 ng/ml) for 7 days. Supernatants were collected, and Ig production was determined by isotype-specific ELISA. Data represent the results of one of the two independent experiments and are means of triplicated samples±SEM. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Effect of LF, RA, and TGF-β1 on Ig secretion by mouse peritoneal B cells. Mouse whole peritoneal B cells were stimulated with LPS (12.5 µg/ml), RA (25 nM), LF (60 µg/ml), and TGF-β1 (0.2 ng/ml) for 7 days. Supernatants were collected, and Ig production was determined by isotype-specific ELISA. Data are means of triplicate samples±SEM. * p
    Figure Legend Snippet: Effect of LF, RA, and TGF-β1 on Ig secretion by mouse peritoneal B cells. Mouse whole peritoneal B cells were stimulated with LPS (12.5 µg/ml), RA (25 nM), LF (60 µg/ml), and TGF-β1 (0.2 ng/ml) for 7 days. Supernatants were collected, and Ig production was determined by isotype-specific ELISA. Data are means of triplicate samples±SEM. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    11) Product Images from "Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels"

    Article Title: Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00015.2014

    Subcellular localization of Smad3 in HFL-1 cells. After transfection of MMP-9 siRNA or control siRNA, human lung fibroblasts were stimulated with TGF-β1 for 30 min followed by immunostaining for Smad3. Nuclear counterstaining was performed with
    Figure Legend Snippet: Subcellular localization of Smad3 in HFL-1 cells. After transfection of MMP-9 siRNA or control siRNA, human lung fibroblasts were stimulated with TGF-β1 for 30 min followed by immunostaining for Smad3. Nuclear counterstaining was performed with

    Techniques Used: Transfection, Immunostaining

    12) Product Images from "Conversion of Peripheral CD4+CD25− Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-? Induction of Transcription Factor Foxp3"

    Article Title: Conversion of Peripheral CD4+CD25− Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-? Induction of Transcription Factor Foxp3

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20030152

    Phenotype of TGF-β–induced anergic/suppressor T cells. (A–D) DO11.10 TCR transgenic spleen cells were stimulated with OVA in the absence (OVA + Med) and presence of TGF-β (OVA + TGF-β) for 7 d. CD4 + T cells were purified and stained with PE–anti-CD4 and FITC–anti-CD25, or FITC–anti-CD45RB. CD4 + T cells ( > 98% KJ1-26 + ) were gated, and histogram profiles of cell size on FSC (A) and CD25 (B) are displayed. Profile of dual CD4 and CD45RB expression (C) CD4 + T cells purified at day 7 after the primary cultures were rested in complete DMEM for 56 h. Viable CD4 + T cells were stained with FITC-anti–KJ1-26 and intracellular PE-anti–CTLA-4. (D) TGF-β–induced anergic/suppressor T cells express membrane-bound TGF-β (E and F). B6 CD4 + CD25 − T cells were cultured with anti-CD3 and APCs in the absence (panels a and b, αCD3 + Med) or presence of TGF-β (panels c and d, αCD3 + TGF-β) for 3 d. After extensive washes, cells were stained with FITC–anti-CD25 and biotinylated chicken anti–TGF-β1, followed by streptavidin-PE. Cells were analyzed on flow cytometry and under immunofluorescence microscopy.
    Figure Legend Snippet: Phenotype of TGF-β–induced anergic/suppressor T cells. (A–D) DO11.10 TCR transgenic spleen cells were stimulated with OVA in the absence (OVA + Med) and presence of TGF-β (OVA + TGF-β) for 7 d. CD4 + T cells were purified and stained with PE–anti-CD4 and FITC–anti-CD25, or FITC–anti-CD45RB. CD4 + T cells ( > 98% KJ1-26 + ) were gated, and histogram profiles of cell size on FSC (A) and CD25 (B) are displayed. Profile of dual CD4 and CD45RB expression (C) CD4 + T cells purified at day 7 after the primary cultures were rested in complete DMEM for 56 h. Viable CD4 + T cells were stained with FITC-anti–KJ1-26 and intracellular PE-anti–CTLA-4. (D) TGF-β–induced anergic/suppressor T cells express membrane-bound TGF-β (E and F). B6 CD4 + CD25 − T cells were cultured with anti-CD3 and APCs in the absence (panels a and b, αCD3 + Med) or presence of TGF-β (panels c and d, αCD3 + TGF-β) for 3 d. After extensive washes, cells were stained with FITC–anti-CD25 and biotinylated chicken anti–TGF-β1, followed by streptavidin-PE. Cells were analyzed on flow cytometry and under immunofluorescence microscopy.

    Techniques Used: Transgenic Assay, Purification, Staining, Expressing, Cell Culture, Flow Cytometry, Cytometry, Immunofluorescence, Microscopy

    TGF-β and TCR costimulation induces Foxp3 expression in CD4 + CD25 − naive responder T cells. (A) B6 spleen cells were sorted into CD4 + CD25 − (CD25 − ) and T reg (CD25 + ) populations. cDNA from each population was subjected to nonsaturating PCR using Foxp3 or HPRT-specific primers, and data are presented as Foxp3 /HPRT ratio. (B) CD25 − cells were cultured with medium or 2 ng/ml TGF-β1 (24 h) or stimulated with platebound anti-CD3 and soluble anti-CD28 in the absence or presence of TGF-β1 (72 h) and assessed for the expression of Foxp3 by RT-PCR. (C) CD25 − cells were activated with soluble anti-CD3 and APCs with or without TGF-β for 3 d and assessed for Foxp3 expression. (D) Dose dependence of TGF-β and failure of IL-10 on Foxp3 induction in CD25 − naive T cells. CD25 − cells were cultured as in B in the presence of indicated concentrations of TGF-β or recombinant murine IL-10. (E) Both TGF-β and IL-10 failed to further enhance Foxp3 expression in T reg . Freshly isolated T reg were activated with platebound anti-CD3, soluble anti-CD28, and IL-2 (100 U/ml) with or without TGF-β or IL-10, and Foxp3 expression was assessed by RT-PCR. (F and G) Flow cytometry analysis (F) and Foxp3 expression (G) of CD25 + or CD25 − T cells sorted from TGF-β– and anti-CD3–costimulated naive CD25 − T cells (day 7). (H) No Foxp3 expression in both CD25 + and CD25 − subsets purified from control (anti-CD3 only) stimulated CD25 − naive cells (day 7). The data in the figure are representative of at least three experiments.
    Figure Legend Snippet: TGF-β and TCR costimulation induces Foxp3 expression in CD4 + CD25 − naive responder T cells. (A) B6 spleen cells were sorted into CD4 + CD25 − (CD25 − ) and T reg (CD25 + ) populations. cDNA from each population was subjected to nonsaturating PCR using Foxp3 or HPRT-specific primers, and data are presented as Foxp3 /HPRT ratio. (B) CD25 − cells were cultured with medium or 2 ng/ml TGF-β1 (24 h) or stimulated with platebound anti-CD3 and soluble anti-CD28 in the absence or presence of TGF-β1 (72 h) and assessed for the expression of Foxp3 by RT-PCR. (C) CD25 − cells were activated with soluble anti-CD3 and APCs with or without TGF-β for 3 d and assessed for Foxp3 expression. (D) Dose dependence of TGF-β and failure of IL-10 on Foxp3 induction in CD25 − naive T cells. CD25 − cells were cultured as in B in the presence of indicated concentrations of TGF-β or recombinant murine IL-10. (E) Both TGF-β and IL-10 failed to further enhance Foxp3 expression in T reg . Freshly isolated T reg were activated with platebound anti-CD3, soluble anti-CD28, and IL-2 (100 U/ml) with or without TGF-β or IL-10, and Foxp3 expression was assessed by RT-PCR. (F and G) Flow cytometry analysis (F) and Foxp3 expression (G) of CD25 + or CD25 − T cells sorted from TGF-β– and anti-CD3–costimulated naive CD25 − T cells (day 7). (H) No Foxp3 expression in both CD25 + and CD25 − subsets purified from control (anti-CD3 only) stimulated CD25 − naive cells (day 7). The data in the figure are representative of at least three experiments.

    Techniques Used: Expressing, Polymerase Chain Reaction, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Recombinant, Isolation, Flow Cytometry, Cytometry, Purification

    13) Product Images from "Smad2 is Involved in Aggregatibacter actinomycetemcomitans-induced Apoptosis"

    Article Title: Smad2 is Involved in Aggregatibacter actinomycetemcomitans-induced Apoptosis

    Journal: Journal of Dental Research

    doi: 10.1177/0022034514550041

    Aa Y4 activated the TGF-β type I receptor. (A) Representative Western blotting of Aa Y4- and TGF-β1-stimulated phosphorylation of serine in TGF-βRI and also of total TGF-βRI after the immunoprecipitation (IP) of TGF-βRI. Lane 1, basal; lane 2, Aa Y4; lane 3, 10 ng/mL TGF-β1. (B) Densitometric analysis of the Aa Y4- or TGF-β1-stimulated phosphorylation of serine in TGF-βRI. OBA9 cells were incubated for 1 hr with Aa Y4 or 10 ng/mL TGF-β1 as a control. TGF-βRI was immunoprecipitated from cell lysates and subjected to Western blotting analysis of the phosphorylation of serine. Blots were re-probed for total TGF-βRI levels. Results are expressed as a percentage above basal levels in relative densitometric units normalized to total TGF-βRI levels. Band densities were quantified through densitometric scanning of each band with the National Institutes of Health Image J software. Values represent the mean ± SD of 3 cultures. ** p
    Figure Legend Snippet: Aa Y4 activated the TGF-β type I receptor. (A) Representative Western blotting of Aa Y4- and TGF-β1-stimulated phosphorylation of serine in TGF-βRI and also of total TGF-βRI after the immunoprecipitation (IP) of TGF-βRI. Lane 1, basal; lane 2, Aa Y4; lane 3, 10 ng/mL TGF-β1. (B) Densitometric analysis of the Aa Y4- or TGF-β1-stimulated phosphorylation of serine in TGF-βRI. OBA9 cells were incubated for 1 hr with Aa Y4 or 10 ng/mL TGF-β1 as a control. TGF-βRI was immunoprecipitated from cell lysates and subjected to Western blotting analysis of the phosphorylation of serine. Blots were re-probed for total TGF-βRI levels. Results are expressed as a percentage above basal levels in relative densitometric units normalized to total TGF-βRI levels. Band densities were quantified through densitometric scanning of each band with the National Institutes of Health Image J software. Values represent the mean ± SD of 3 cultures. ** p

    Techniques Used: Western Blot, Immunoprecipitation, Incubation, Software

    14) Product Images from "Alteration of Wnt5a expression and of the non-canonical Wnt/PCP and Wnt/PKC-Ca2+ pathways in human osteoarthritis osteoblasts"

    Article Title: Alteration of Wnt5a expression and of the non-canonical Wnt/PCP and Wnt/PKC-Ca2+ pathways in human osteoarthritis osteoblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0180711

    Reciprocal regulation of Wnt5a and TGF-β1 expression in OA osteoblasts. Confluent OA osteoblasts were treated with either rhWnt5a or siWnt5a in order to measure the expression of TGF-β1. Conversely, OA osteoblasts were treated with either rhTGF-β1 or siTGF-β1 prior to the measure of Wnt5a expression. Gene expression was measured by RT-qPCR. A) Expression of TGF-β1 in response to siWnt5a treatement (n = 4); B) Expression of TGF-β1 in response to rhWnt5a (n = 3); C) Expression of TGF-β1 in response to siTGF-β1 treatment (n = 6); D) Expression of Wnt5a in response to rhTGF-β1 (n = 3); E) Expression of Wnt5a in response to siTGF-β1 (n = 4).
    Figure Legend Snippet: Reciprocal regulation of Wnt5a and TGF-β1 expression in OA osteoblasts. Confluent OA osteoblasts were treated with either rhWnt5a or siWnt5a in order to measure the expression of TGF-β1. Conversely, OA osteoblasts were treated with either rhTGF-β1 or siTGF-β1 prior to the measure of Wnt5a expression. Gene expression was measured by RT-qPCR. A) Expression of TGF-β1 in response to siWnt5a treatement (n = 4); B) Expression of TGF-β1 in response to rhWnt5a (n = 3); C) Expression of TGF-β1 in response to siTGF-β1 treatment (n = 6); D) Expression of Wnt5a in response to rhTGF-β1 (n = 3); E) Expression of Wnt5a in response to siTGF-β1 (n = 4).

    Techniques Used: Expressing, Quantitative RT-PCR

    15) Product Images from "The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-?, increases podocyte motility and albumin permeability"

    Article Title: The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-?, increases podocyte motility and albumin permeability

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.90642.2008

    MCP-1 stimulates podocyte motility as evaluated by a scratch-wound assay. A : in the counting of the number of cells that had repopulated a consistently defined area of the scratch, MCP-1 was seen to significantly stimulate podocyte migration at all time points, resulting in quicker wound closure. A similar effect was seen with TGF-β1 treatment. Both MCP-1- and TGF-β1-induced motility increases were prevented by CCR2 inhibition with RS102895 ( n = 4). * P
    Figure Legend Snippet: MCP-1 stimulates podocyte motility as evaluated by a scratch-wound assay. A : in the counting of the number of cells that had repopulated a consistently defined area of the scratch, MCP-1 was seen to significantly stimulate podocyte migration at all time points, resulting in quicker wound closure. A similar effect was seen with TGF-β1 treatment. Both MCP-1- and TGF-β1-induced motility increases were prevented by CCR2 inhibition with RS102895 ( n = 4). * P

    Techniques Used: Scratch Wound Assay Assay, Migration, Inhibition

    TGF-β stimulates monocyte chemoattractant protein-1 (MCP-1) expression. A : cultured, differentiated mouse podocytes were treated with 2 ng/ml of recombinant transforming growth factor (TGF)-β1 for 48 h. Compared with control, TGF-β1 markedly increased MCP-1 production as measured by ELISA of cell lysate. The TGF-β-stimulated MCP-1 production was significantly blunted by concurrent treatment with 1 μM SB431542 ( n = 3). * P
    Figure Legend Snippet: TGF-β stimulates monocyte chemoattractant protein-1 (MCP-1) expression. A : cultured, differentiated mouse podocytes were treated with 2 ng/ml of recombinant transforming growth factor (TGF)-β1 for 48 h. Compared with control, TGF-β1 markedly increased MCP-1 production as measured by ELISA of cell lysate. The TGF-β-stimulated MCP-1 production was significantly blunted by concurrent treatment with 1 μM SB431542 ( n = 3). * P

    Techniques Used: Expressing, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay

    Phosphatidylinositol 3-kinase (PI3K) mediates TGF-β1-stimulated MCP-1. A : a specific inhibitor of PI3K, LY294002 (25 μM), completely inhibited TGF-β-stimulated MCP-1 production by cultured podocytes ( n = 3). The modest decrease in MCP-1 due to LY294002 alone was not significantly different from control. * P
    Figure Legend Snippet: Phosphatidylinositol 3-kinase (PI3K) mediates TGF-β1-stimulated MCP-1. A : a specific inhibitor of PI3K, LY294002 (25 μM), completely inhibited TGF-β-stimulated MCP-1 production by cultured podocytes ( n = 3). The modest decrease in MCP-1 due to LY294002 alone was not significantly different from control. * P

    Techniques Used: Cell Culture

    16) Product Images from "The aryl hydrocarbon receptor agonist benzo(a)pyrene reactivates LINE-1 in HepG2 cells through canonical TGF-β1 signaling: implications in hepatocellular carcinogenesis"

    Article Title: The aryl hydrocarbon receptor agonist benzo(a)pyrene reactivates LINE-1 in HepG2 cells through canonical TGF-β1 signaling: implications in hepatocellular carcinogenesis

    Journal: American Journal of Cancer Research

    doi:

    Induction of L1 and TGF-β1 expression by BaP is dependent upon AhR signaling. HepG2 cells were stimulated with: (A) different concentrations of BaP or an equivalent volume of DMSO for 24 h, or (B) 1.5 μM BaP or DMSO (0.5%) for different
    Figure Legend Snippet: Induction of L1 and TGF-β1 expression by BaP is dependent upon AhR signaling. HepG2 cells were stimulated with: (A) different concentrations of BaP or an equivalent volume of DMSO for 24 h, or (B) 1.5 μM BaP or DMSO (0.5%) for different

    Techniques Used: Expressing

    Expression of L1 proteins correlates with the activation of TGF-β1 signaling in liver tumors. Whole extracts of human liver hepatocellular carcinoma stages I, II, III, IV, and normal tissue were analyzed as follows: A. Immunoblotting for L1 protein
    Figure Legend Snippet: Expression of L1 proteins correlates with the activation of TGF-β1 signaling in liver tumors. Whole extracts of human liver hepatocellular carcinoma stages I, II, III, IV, and normal tissue were analyzed as follows: A. Immunoblotting for L1 protein

    Techniques Used: Expressing, Activation Assay

    TGF-β1 induces L1 mRNA expression in HepG2 cells. Cells were stimulated for different times by addition of 10 ng/ml TGF-β1. Total RNA was isolated, and 1 μg of RNA subjected to cDNA synthesis. Samples were analyzed via RT-PCR using
    Figure Legend Snippet: TGF-β1 induces L1 mRNA expression in HepG2 cells. Cells were stimulated for different times by addition of 10 ng/ml TGF-β1. Total RNA was isolated, and 1 μg of RNA subjected to cDNA synthesis. Samples were analyzed via RT-PCR using

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

    Activation of TGF-β1 signaling by BaP in coupled to AhR. Cells were treated with 1.5 mM BaP or an equivalent volume of DMSO for different periods of time. A. Whole cell lysates were analyzed by immunoblotting for phospho-SMAD2, total SMAD2, E-Cadherin,
    Figure Legend Snippet: Activation of TGF-β1 signaling by BaP in coupled to AhR. Cells were treated with 1.5 mM BaP or an equivalent volume of DMSO for different periods of time. A. Whole cell lysates were analyzed by immunoblotting for phospho-SMAD2, total SMAD2, E-Cadherin,

    Techniques Used: Activation Assay

    17) 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

    18) Product Images from "Red-COLA1: a human fibroblast reporter cell line for type I collagen transcription"

    Article Title: Red-COLA1: a human fibroblast reporter cell line for type I collagen transcription

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-75683-5

    Anti-fibrotics targeting TGF-β1 signalling decrease m-Cherry expression in reporter cells. Red-COLA1 were co-treated with 10 ng/mL of TGF-β1 and decreasing concentration (10 to 0.3 μM) of (a) Nintedanib, (b) JQ-1, (c) Omapalisib, (d) Halofugione, (e) ICG-001 and (f) Nilotinib. Average percentage of m-Cherry positive nucleus were quantified and calculated, three days post treatment using manufacturer image analysis. Error bars represent SD from 4 sites performed in 6 replicate wells at each concentration.
    Figure Legend Snippet: Anti-fibrotics targeting TGF-β1 signalling decrease m-Cherry expression in reporter cells. Red-COLA1 were co-treated with 10 ng/mL of TGF-β1 and decreasing concentration (10 to 0.3 μM) of (a) Nintedanib, (b) JQ-1, (c) Omapalisib, (d) Halofugione, (e) ICG-001 and (f) Nilotinib. Average percentage of m-Cherry positive nucleus were quantified and calculated, three days post treatment using manufacturer image analysis. Error bars represent SD from 4 sites performed in 6 replicate wells at each concentration.

    Techniques Used: Expressing, Concentration Assay

    Design and generation of Red-COLA1 reporter cell line. (a) TALENs binding sites in COL1A1 genomic locus. TALENs recognize bases + 11 (in red) and + 43 (in blue) base pairs from the start codon (underlined) in endogenous COL1A1 genomic locus. Donor DNA cassette comprises of a nuclear localizing mCherry-reporter (NLS-mCherry) fused to a destabilising proline-glutamate-serine-threonine-rich (PEST) sequence signal, followed by polyA tail (pA) flanked by two homologous recombination arms (5′L and 3′R). (b) Schematic diagram of knock-in allele. Primer binding sites (F1, R1, F2, R2) and their expected band sizes for junctional PCR analysis are denoted with arrows (bottom). (c) Junctional PCR. Amplification of the integrated cassette was detected with 2 independent pair of primers that specifically target endogenous COL1A1 and m-Cherry sequences. Full length gels are presented in Supplementary Fig. 1 e. The primer positions are indicated on the schematic of the targeted COL1A1 locus in (a,b) . (d) Expression of both mCherry and endogenous collagen (FITC stained) in Red-COLA1 reporter cells at 72 h following 10 ng/mL of TGF-β1 stimulation. Red-COLA1 cells immunostained with rabbit antibodies for type I collagen 1 (COL1) and labelled with anti-rabbit conjugated with FITC (COL1-FITC). Image acquisition (10 ×) was performed 3 days post-induction. (e) Representative live images of Red-COLA1 treated with TGF-β1 for 24, 48 and 72 h. (f) Cells were fixed at the end of the experiment and stained with Hoechst 33342 to reveal the nuclei. The intensity levels of both mCherry and nuclei count from 4 sites of 3 independent wells were calculated using the manufacturer’s analysis software and normalised to their respective untreated controls (24 or 72 h). Error bars represent SD from two independent experiments. (g) . Red-COLA1 derived from post FACS-enrichment were cultured for 20 passages and imaged for m-Cherry expression. Images were acquired from fixed cells at 72 h following 10 ng/mL of TGF-β1 stimulation. (h) Quantification of TGF-β1-responsive cells at different cell passages. Percentage of m-Cherry positive cells were calculated by applying a nuclei mask and quantifying the number of m-Cherry positive signal that overlaps with nuclei mask over the total nuclei detected.
    Figure Legend Snippet: Design and generation of Red-COLA1 reporter cell line. (a) TALENs binding sites in COL1A1 genomic locus. TALENs recognize bases + 11 (in red) and + 43 (in blue) base pairs from the start codon (underlined) in endogenous COL1A1 genomic locus. Donor DNA cassette comprises of a nuclear localizing mCherry-reporter (NLS-mCherry) fused to a destabilising proline-glutamate-serine-threonine-rich (PEST) sequence signal, followed by polyA tail (pA) flanked by two homologous recombination arms (5′L and 3′R). (b) Schematic diagram of knock-in allele. Primer binding sites (F1, R1, F2, R2) and their expected band sizes for junctional PCR analysis are denoted with arrows (bottom). (c) Junctional PCR. Amplification of the integrated cassette was detected with 2 independent pair of primers that specifically target endogenous COL1A1 and m-Cherry sequences. Full length gels are presented in Supplementary Fig. 1 e. The primer positions are indicated on the schematic of the targeted COL1A1 locus in (a,b) . (d) Expression of both mCherry and endogenous collagen (FITC stained) in Red-COLA1 reporter cells at 72 h following 10 ng/mL of TGF-β1 stimulation. Red-COLA1 cells immunostained with rabbit antibodies for type I collagen 1 (COL1) and labelled with anti-rabbit conjugated with FITC (COL1-FITC). Image acquisition (10 ×) was performed 3 days post-induction. (e) Representative live images of Red-COLA1 treated with TGF-β1 for 24, 48 and 72 h. (f) Cells were fixed at the end of the experiment and stained with Hoechst 33342 to reveal the nuclei. The intensity levels of both mCherry and nuclei count from 4 sites of 3 independent wells were calculated using the manufacturer’s analysis software and normalised to their respective untreated controls (24 or 72 h). Error bars represent SD from two independent experiments. (g) . Red-COLA1 derived from post FACS-enrichment were cultured for 20 passages and imaged for m-Cherry expression. Images were acquired from fixed cells at 72 h following 10 ng/mL of TGF-β1 stimulation. (h) Quantification of TGF-β1-responsive cells at different cell passages. Percentage of m-Cherry positive cells were calculated by applying a nuclei mask and quantifying the number of m-Cherry positive signal that overlaps with nuclei mask over the total nuclei detected.

    Techniques Used: TALENs, Binding Assay, Sequencing, Homologous Recombination, Knock-In, Polymerase Chain Reaction, Amplification, Expressing, Staining, Software, Derivative Assay, FACS, Cell Culture

    RNAi screen with Red-COLA1 identifies genes regulating TGF-β1 signalling. (a) Workflow of RNAI screen. Transfection reagent (RNAiMAX) was diluted in serum free media prior to dispensing into 384-well plates pre-printed with siRNA targeting genes in the human kinome and phosphatome library. Red-COLA1 cells were trypsinized, resuspended in complete growth media (DMEM, 10% FBS) and then added to wells twenty minutes after siRNA were complexed with the transfection reagent. TGF-β1 were added to wells 3 days post transfection, at a final concentration of 1 ng/mL. Image acquisition was performed 6 days post transfection. (b) Control siRNAs used in the screen. 2 negative control siRNAs, non-targeting siRNA (siNT), Giantin (siGaintin) and 2 positive control siRNAs targeting Smad3 were included in control wells of the screen. Non-specific m-Cherry signals observed after siRNA transfection were denoted by white open arrows. A DAPI mask was applied to quantify nuclear-localized m-Cherry signal (bottom panel) and the percentage of cells positive for nuclear m-Cherry expression above applied threshold was determined. (c) Average percentage of m-Cherry positive nucleus were quantified and calculated using manufacturer image analysis software. Error bars represent SD from 4 sites performed in 4 replicate wells. (d) Box plot of controls (si-Smad3-1, si-Smad3-2, siNT, siGiantin) and samples in the screen. A: All wells. S: Sample wells. C: Controls. (e) Hits identification. Sample wells with percentage of nuclear m-Cherry positive cells at least three standard deviations away (3SD) from the average of siNT (orange) controls were identified as hits. Distribution of other control wells, Giantin and Smad 3–2 are highlighted in yellow and green respectively. (f) Positive regulators of TGF-β1-induced m-Cherry expression. Depletion of genes that down-regulate TGF-β1-induced m-Cherry positive cells are highlighted in red. Some genes previously implicated in the pathway are indicated (ACVRL1, TGFBR1, TRIB3, LYN, LCK) (g) Negative regulators of TGF-β1-induced m-Cherry expression. Depletion of genes that up-regulate TGF-β1-induced m-Cherry cells are highlighted in red. Some genes previously implicated in the pathway are indicated (CHUK1, IKBKE, PICK1).
    Figure Legend Snippet: RNAi screen with Red-COLA1 identifies genes regulating TGF-β1 signalling. (a) Workflow of RNAI screen. Transfection reagent (RNAiMAX) was diluted in serum free media prior to dispensing into 384-well plates pre-printed with siRNA targeting genes in the human kinome and phosphatome library. Red-COLA1 cells were trypsinized, resuspended in complete growth media (DMEM, 10% FBS) and then added to wells twenty minutes after siRNA were complexed with the transfection reagent. TGF-β1 were added to wells 3 days post transfection, at a final concentration of 1 ng/mL. Image acquisition was performed 6 days post transfection. (b) Control siRNAs used in the screen. 2 negative control siRNAs, non-targeting siRNA (siNT), Giantin (siGaintin) and 2 positive control siRNAs targeting Smad3 were included in control wells of the screen. Non-specific m-Cherry signals observed after siRNA transfection were denoted by white open arrows. A DAPI mask was applied to quantify nuclear-localized m-Cherry signal (bottom panel) and the percentage of cells positive for nuclear m-Cherry expression above applied threshold was determined. (c) Average percentage of m-Cherry positive nucleus were quantified and calculated using manufacturer image analysis software. Error bars represent SD from 4 sites performed in 4 replicate wells. (d) Box plot of controls (si-Smad3-1, si-Smad3-2, siNT, siGiantin) and samples in the screen. A: All wells. S: Sample wells. C: Controls. (e) Hits identification. Sample wells with percentage of nuclear m-Cherry positive cells at least three standard deviations away (3SD) from the average of siNT (orange) controls were identified as hits. Distribution of other control wells, Giantin and Smad 3–2 are highlighted in yellow and green respectively. (f) Positive regulators of TGF-β1-induced m-Cherry expression. Depletion of genes that down-regulate TGF-β1-induced m-Cherry positive cells are highlighted in red. Some genes previously implicated in the pathway are indicated (ACVRL1, TGFBR1, TRIB3, LYN, LCK) (g) Negative regulators of TGF-β1-induced m-Cherry expression. Depletion of genes that up-regulate TGF-β1-induced m-Cherry cells are highlighted in red. Some genes previously implicated in the pathway are indicated (CHUK1, IKBKE, PICK1).

    Techniques Used: Transfection, Concentration Assay, Negative Control, Positive Control, Expressing, Software

    Red-COLA1 cells accurately depict changes in COL1A1 activation. (a) Relative mRNA expression of COL1A1 and mCherry at 24, 48, or 72 h after stimulation with10 ng/mL TGF-β1. Relative expression of the target genes was calculated with reference to the untreated control. β-actin was used as an internal control. Error bars represent SD of the average of two independent experiments performed in triplicates. (b) Total cell lysates (TCL) from cells treated with TGF-β1 were probed for mCherry and pro-collagen (Pro-Col I). β-tubulin was included as a loading control. Full length blots are presented in Supplementary Fig. 3 (c) . Intensity of bands corresponding to type I collagen or m-Cherry was quantified using ImageJ and normalised with β-tubulin and expressed relative to respective 0 h control. Error bars showed standard deviation of fold-change averaged from 3 independent experiments. (d) Representative images of Red-COLA1 fibroblasts stimulated with 1.25, 2.5, and 5 ng/mL of TGF-β1. Image acquisition (10 ×) was performed 24 or 72 h post-induction with a high-content imager (ImageXpress) in live cells. (e) The intensity levels of both mCherry and nuclei count from 4 sites of 3 independent wells were calculated using the manufacturer’s analysis software and normalised to their respective untreated controls (24 or 72hours). Error bars represent SD from two independent experiments. (f) Representative images from untreated or TGF-β1 stimulated Red-COLA1 fibroblasts co-treated with 0.1 ng/mL of TNF or 1 µM TGF-β1 receptor inhibitor, SB431542. (g) Quantification of immunofluorescence in (f) . The intensity levels of both mCherry and type I collagen-FITC per cell, from 4 sites of 3 independent wells were calculated and normalised to untreated controls. Error bars represent SD from three independent experiments. (h) Untreated and TGF-β1-treated (72 h) Red-COL1A1 cells immunostained with rabbit antibodies for type I collagen 1 (COL1) and labelled with anti-rabbit conjugated with FITC (COL1-FITC). (i) The total intensity levels of both mCherry and type I collagen-FITC per nuclei (DAPI), 5 independent wells were calculated using the manufacturer’s analysis software and normalised to untreated controls. Error bars represent SD.
    Figure Legend Snippet: Red-COLA1 cells accurately depict changes in COL1A1 activation. (a) Relative mRNA expression of COL1A1 and mCherry at 24, 48, or 72 h after stimulation with10 ng/mL TGF-β1. Relative expression of the target genes was calculated with reference to the untreated control. β-actin was used as an internal control. Error bars represent SD of the average of two independent experiments performed in triplicates. (b) Total cell lysates (TCL) from cells treated with TGF-β1 were probed for mCherry and pro-collagen (Pro-Col I). β-tubulin was included as a loading control. Full length blots are presented in Supplementary Fig. 3 (c) . Intensity of bands corresponding to type I collagen or m-Cherry was quantified using ImageJ and normalised with β-tubulin and expressed relative to respective 0 h control. Error bars showed standard deviation of fold-change averaged from 3 independent experiments. (d) Representative images of Red-COLA1 fibroblasts stimulated with 1.25, 2.5, and 5 ng/mL of TGF-β1. Image acquisition (10 ×) was performed 24 or 72 h post-induction with a high-content imager (ImageXpress) in live cells. (e) The intensity levels of both mCherry and nuclei count from 4 sites of 3 independent wells were calculated using the manufacturer’s analysis software and normalised to their respective untreated controls (24 or 72hours). Error bars represent SD from two independent experiments. (f) Representative images from untreated or TGF-β1 stimulated Red-COLA1 fibroblasts co-treated with 0.1 ng/mL of TNF or 1 µM TGF-β1 receptor inhibitor, SB431542. (g) Quantification of immunofluorescence in (f) . The intensity levels of both mCherry and type I collagen-FITC per cell, from 4 sites of 3 independent wells were calculated and normalised to untreated controls. Error bars represent SD from three independent experiments. (h) Untreated and TGF-β1-treated (72 h) Red-COL1A1 cells immunostained with rabbit antibodies for type I collagen 1 (COL1) and labelled with anti-rabbit conjugated with FITC (COL1-FITC). (i) The total intensity levels of both mCherry and type I collagen-FITC per nuclei (DAPI), 5 independent wells were calculated using the manufacturer’s analysis software and normalised to untreated controls. Error bars represent SD.

    Techniques Used: Activation Assay, Expressing, Standard Deviation, Software, Immunofluorescence

    19) Product Images from "Estrogen receptor-α-miR-1271-SNAI2 feedback loop regulates transforming growth factor-β-induced breast cancer progression"

    Article Title: Estrogen receptor-α-miR-1271-SNAI2 feedback loop regulates transforming growth factor-β-induced breast cancer progression

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1112-4

    Estrogen reverses TGF-β-induced EMT in a miR-1271 dependent manner. a The expression of miR-1271 in T47D cells after treatment with TGF-β1 at indicated concentration. b Transwell invasion assay of miR-1271-depleted T47D or control cells with TGF-β1 or/and E2 treatment. c Luciferase reporter analysis of TGF-β signaling activity in cells as in (B). d Localization of SMAD2 in cells as in (B) as determined by immunofluorescence staining. e The expression of pSMAD2 in cells as in (A) by western blotting. f The expression of SNAI1/2, TWIST1 and ZEB1 in cells as in (A) as determined by RT-qPCR. * P
    Figure Legend Snippet: Estrogen reverses TGF-β-induced EMT in a miR-1271 dependent manner. a The expression of miR-1271 in T47D cells after treatment with TGF-β1 at indicated concentration. b Transwell invasion assay of miR-1271-depleted T47D or control cells with TGF-β1 or/and E2 treatment. c Luciferase reporter analysis of TGF-β signaling activity in cells as in (B). d Localization of SMAD2 in cells as in (B) as determined by immunofluorescence staining. e The expression of pSMAD2 in cells as in (A) by western blotting. f The expression of SNAI1/2, TWIST1 and ZEB1 in cells as in (A) as determined by RT-qPCR. * P

    Techniques Used: Expressing, Concentration Assay, Transwell Invasion Assay, Luciferase, Activity Assay, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

    20) Product Images from "FAD104, a regulator of adipogenesis, is a novel suppressor of TGF-β–mediated EMT in cervical cancer cells"

    Article Title: FAD104, a regulator of adipogenesis, is a novel suppressor of TGF-β–mediated EMT in cervical cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16555-3

    FAD104 overexpression attenuates TGF-β–mediated EMT in HeLa cells. ( A ) The effect of FAD104 overexpression on the expression level of EMT-related genes. HeLa cells were infected with FAD104 or LacZ, and treated with 1 ng/mL TGF-β1 for 72 h. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities of the proteins were quantified using NIH-Image software. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p
    Figure Legend Snippet: FAD104 overexpression attenuates TGF-β–mediated EMT in HeLa cells. ( A ) The effect of FAD104 overexpression on the expression level of EMT-related genes. HeLa cells were infected with FAD104 or LacZ, and treated with 1 ng/mL TGF-β1 for 72 h. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities of the proteins were quantified using NIH-Image software. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p

    Techniques Used: Over Expression, Expressing, Infection, Western Blot, Software, Standard Deviation

    FAD104 expression is elevated during TGF-β–mediated EMT in NMuMG cells. NMuMG cells were treated with 5 ng/mL TGF-β1 or vehicle for 48 h. ( A ) Morphological changes in NMuMG cells after treatment with TGF-β1. F-actin was visualized by TRITC-conjugated phalloidin. ( B ) Expression of the epithelial marker ZO-1 and mesenchymal marker N-cadherin after treatment with TGF-β1. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. ( C ) qPCR analysis of fad104 expression in NMuMG cells treated with TGF-β1. The expression level of fad104 was normalized with 18 S rRNA expression. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p
    Figure Legend Snippet: FAD104 expression is elevated during TGF-β–mediated EMT in NMuMG cells. NMuMG cells were treated with 5 ng/mL TGF-β1 or vehicle for 48 h. ( A ) Morphological changes in NMuMG cells after treatment with TGF-β1. F-actin was visualized by TRITC-conjugated phalloidin. ( B ) Expression of the epithelial marker ZO-1 and mesenchymal marker N-cadherin after treatment with TGF-β1. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. ( C ) qPCR analysis of fad104 expression in NMuMG cells treated with TGF-β1. The expression level of fad104 was normalized with 18 S rRNA expression. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p

    Techniques Used: Expressing, Marker, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    FAD104 positively regulates phosphorylation level of Smad1/5/8 with TGF-β1 treatment in HeLa cells. ( A ) Phosphorylation levels of Smad1/5/8 in fad104 knockdown HeLa cells. HeLa cells were transfected with siRNA targeting fad104 (sifad104-A) and treated with 1 ng/mL TGF-β1 for 30 min. ( B ) Phosphorylation levels of Smad1/5/8 in HeLa cells overexpressing FAD104. HeLa cells were infected with FAD104 and treated with 1 ng/mL TGF-β1 for 30 min. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities from phospho-Smad1/5/8, total Smad1/5/8, and β-actin were quantified using NIH-Image software. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p
    Figure Legend Snippet: FAD104 positively regulates phosphorylation level of Smad1/5/8 with TGF-β1 treatment in HeLa cells. ( A ) Phosphorylation levels of Smad1/5/8 in fad104 knockdown HeLa cells. HeLa cells were transfected with siRNA targeting fad104 (sifad104-A) and treated with 1 ng/mL TGF-β1 for 30 min. ( B ) Phosphorylation levels of Smad1/5/8 in HeLa cells overexpressing FAD104. HeLa cells were infected with FAD104 and treated with 1 ng/mL TGF-β1 for 30 min. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities from phospho-Smad1/5/8, total Smad1/5/8, and β-actin were quantified using NIH-Image software. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p

    Techniques Used: Transfection, Infection, Western Blot, Software, Standard Deviation

    Fad104 knockdown enhances TGF-β–mediated EMT in HeLa cells. ( A ) Knockdown efficiency of fad104 in HeLa cells. HeLa cells were transfected with siRNA targeting fad104 (sifad104-A) and treated with 5 ng/mL TGF-β1. Luciferase siRNA was used as a control. β-Actin expression was used as a loading control. ( B ) Morphological changes in HeLa cells transfected with fad104 siRNA. Cells were treated with 5 ng/mL TGF-β1 for 72 h. F-actin was visualized by TRITC-conjugated phalloidin. Scale bars represent 100 μm. ( C ) Quantitative analysis of cell morphology of HeLa cells in ( B ). The lengths of the major and minor cell axes were measured using NIH-Image software. The ratios of the major to minor axes of cells were used to determine the degree of elongated cell morphology. For each experiment, over 20 cells in each condition were measured. Each column represents the mean with standard deviation. ( D ) qPCR analysis of fibronectin, snail , and slug expression in fad104 knockdown cells. Cells were treated with 1 ng/mL TGF-β1 for 72 h. Expression levels of fibronectin, snail , and slug were normalized with 18 S rRNA expression. Each column represents the mean with standard deviation (n = 3). ( E ) Protein expression of fibronectin, Snail, and Slug in fad104 knockdown cells. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities of the proteins were quantified using NIH-Image software. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p
    Figure Legend Snippet: Fad104 knockdown enhances TGF-β–mediated EMT in HeLa cells. ( A ) Knockdown efficiency of fad104 in HeLa cells. HeLa cells were transfected with siRNA targeting fad104 (sifad104-A) and treated with 5 ng/mL TGF-β1. Luciferase siRNA was used as a control. β-Actin expression was used as a loading control. ( B ) Morphological changes in HeLa cells transfected with fad104 siRNA. Cells were treated with 5 ng/mL TGF-β1 for 72 h. F-actin was visualized by TRITC-conjugated phalloidin. Scale bars represent 100 μm. ( C ) Quantitative analysis of cell morphology of HeLa cells in ( B ). The lengths of the major and minor cell axes were measured using NIH-Image software. The ratios of the major to minor axes of cells were used to determine the degree of elongated cell morphology. For each experiment, over 20 cells in each condition were measured. Each column represents the mean with standard deviation. ( D ) qPCR analysis of fibronectin, snail , and slug expression in fad104 knockdown cells. Cells were treated with 1 ng/mL TGF-β1 for 72 h. Expression levels of fibronectin, snail , and slug were normalized with 18 S rRNA expression. Each column represents the mean with standard deviation (n = 3). ( E ) Protein expression of fibronectin, Snail, and Slug in fad104 knockdown cells. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities of the proteins were quantified using NIH-Image software. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p

    Techniques Used: Transfection, Luciferase, Expressing, Software, Standard Deviation, Real-time Polymerase Chain Reaction, Western Blot

    Knockdown of fad104 expression enhances the migration of HeLa cells undergoing TGF-β–mediated EMT. HeLa cells were transfected with siRNA targeting fad104 (sifad104-A) and treated with 1 ng/mL TGF-β1 for 72 h. ( A ) Whole-cell lysates prepared from HeLa cells undergoing EMT were subjected to Western blot analysis. β-Actin was used as a loading control. Arrowhead shows nonspecific bands. ( B ) Cells undergoing EMT were plated in the upper chamber of the filters coated with fibronectin. Cells that migrated to the underside of the transwell insert were measured after 24 h. Representative images of migrated cells were shown. ( C ) The mean number of migrated cells in the field was calculated. Each column represents the mean with standard deviation (n = 5). ( D ) Cells undergoing EMT were plated in culture plates. After 24 h, cells were trypsinized and counted. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p
    Figure Legend Snippet: Knockdown of fad104 expression enhances the migration of HeLa cells undergoing TGF-β–mediated EMT. HeLa cells were transfected with siRNA targeting fad104 (sifad104-A) and treated with 1 ng/mL TGF-β1 for 72 h. ( A ) Whole-cell lysates prepared from HeLa cells undergoing EMT were subjected to Western blot analysis. β-Actin was used as a loading control. Arrowhead shows nonspecific bands. ( B ) Cells undergoing EMT were plated in the upper chamber of the filters coated with fibronectin. Cells that migrated to the underside of the transwell insert were measured after 24 h. Representative images of migrated cells were shown. ( C ) The mean number of migrated cells in the field was calculated. Each column represents the mean with standard deviation (n = 5). ( D ) Cells undergoing EMT were plated in culture plates. After 24 h, cells were trypsinized and counted. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p

    Techniques Used: Expressing, Migration, Transfection, Western Blot, Standard Deviation

    FAD104 expression is elevated during TGF-β–mediated EMT in HeLa cells. HeLa cells were treated with 5 ng/mL TGF-β1 or vehicle for 72 h. ( A ) Morphological changes of HeLa cells after treatment with TGF-β1. F-actin was visualized by TRITC-conjugated phalloidin. ( B ) The expression of the epithelial marker ZO-1 and mesenchymal marker Fibronectin after treatment with TGF-β1. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. ( C ) qPCR analysis of fad104 expression in HeLa cells treated with TGF-β1. The expression level of fad104 was normalized with 18 S rRNA expression. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p
    Figure Legend Snippet: FAD104 expression is elevated during TGF-β–mediated EMT in HeLa cells. HeLa cells were treated with 5 ng/mL TGF-β1 or vehicle for 72 h. ( A ) Morphological changes of HeLa cells after treatment with TGF-β1. F-actin was visualized by TRITC-conjugated phalloidin. ( B ) The expression of the epithelial marker ZO-1 and mesenchymal marker Fibronectin after treatment with TGF-β1. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. ( C ) qPCR analysis of fad104 expression in HeLa cells treated with TGF-β1. The expression level of fad104 was normalized with 18 S rRNA expression. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p

    Techniques Used: Expressing, Marker, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    FAD104 negatively regulates phosphorylation level of Smad3 with TGF-β1 treatment in HeLa cells. ( A ) Phosphorylation levels of Smad3 in fad104 knockdown HeLa cells. HeLa cells were transfected with siRNA targeting fad104 (sifad104-A) and treated with 1 ng/mL TGF-β1 for 6 h. ( B ) Phosphorylation levels of Smad3 in HeLa cells overexpressing FAD104. HeLa cells were infected with FAD104 and treated with 1 ng/mL TGF-β1 for 6 h. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities from phospho-Smad3, total Smad3, and β-actin were quantified using NIH-Image software. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p
    Figure Legend Snippet: FAD104 negatively regulates phosphorylation level of Smad3 with TGF-β1 treatment in HeLa cells. ( A ) Phosphorylation levels of Smad3 in fad104 knockdown HeLa cells. HeLa cells were transfected with siRNA targeting fad104 (sifad104-A) and treated with 1 ng/mL TGF-β1 for 6 h. ( B ) Phosphorylation levels of Smad3 in HeLa cells overexpressing FAD104. HeLa cells were infected with FAD104 and treated with 1 ng/mL TGF-β1 for 6 h. Whole-cell lysates were subjected to Western blot analysis and β-actin was used as a loading control. Signal intensities from phospho-Smad3, total Smad3, and β-actin were quantified using NIH-Image software. Each column represents the mean with standard deviation (n = 3). Significant differences are indicated as ** p

    Techniques Used: Transfection, Infection, Western Blot, Software, Standard Deviation

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    Recombinant:

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    R&D Systems tgf β1
    Expressions of <t>TGF-β1</t> and IL-10 protein
    Tgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tgf β1/product/R&D Systems
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    R&D Systems recombinant human tgf β1
    The transforming growth factor <t>(TGF)-β</t> pathway is activated during culture of mouse ATII cells. ATII cells were freshly isolated (D0) or cultured for 7 days on tissue culture plastic. The mRNA expression levels of <t>Tgf-β1</t> , Tgf-β2
    Recombinant Human Tgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expressions of TGF-β1 and IL-10 protein

    Journal: Annals of Dermatology

    Article Title: The Expressions of TGF-?1 and IL-10 in Cultured Fibroblasts after ALA-IPL Photodynamic Treatment

    doi: 10.5021/ad.2011.23.1.19

    Figure Lengend Snippet: Expressions of TGF-β1 and IL-10 protein

    Article Snippet: The TGF-β1 and IL-10 ELISA kits and the monoclonal antibodies against these molecules were obtained from R & D systems (USA).

    Techniques:

    Receptor CD36 is required for apoptotic cells induced TGF-β1 mRNA and protein expression. A, CD36 protein expression in RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h was analyzed by Western blotting with anti-CD36 (MF3) antibody, and band density was normalized to GAPDH. B, RAWTβRII cells transfected with CD36-target siRNA or control vehicle (Ctr siRNA) for 24 h were stimulated with apoptotic or viable Jurkat cells. C, Apoptotic Jurkat cells were pretreated with annexin V for 45 min and incubated with RAWTβRII transfected with CD36-target siRNA or control siRNA (Ctr siRNA). TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. D, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) were treated with either LPS (25 ng/ml) or PMA (50 nM). The expression of TGF-β1 mRNA was analyzed as above. Values represent means ± SD of five separate experiments. ns, no significant; *, P

    Journal: PLoS ONE

    Article Title: Induction of TGF-?1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

    doi: 10.1371/journal.pone.0072772

    Figure Lengend Snippet: Receptor CD36 is required for apoptotic cells induced TGF-β1 mRNA and protein expression. A, CD36 protein expression in RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h was analyzed by Western blotting with anti-CD36 (MF3) antibody, and band density was normalized to GAPDH. B, RAWTβRII cells transfected with CD36-target siRNA or control vehicle (Ctr siRNA) for 24 h were stimulated with apoptotic or viable Jurkat cells. C, Apoptotic Jurkat cells were pretreated with annexin V for 45 min and incubated with RAWTβRII transfected with CD36-target siRNA or control siRNA (Ctr siRNA). TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. D, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) were treated with either LPS (25 ng/ml) or PMA (50 nM). The expression of TGF-β1 mRNA was analyzed as above. Values represent means ± SD of five separate experiments. ns, no significant; *, P

    Article Snippet: Antibodies and reagents TGF-β1 was from R & D Systems (Minneapolis, MN).

    Techniques: Expressing, Transfection, Western Blot, Incubation

    Both Lyn kinase and ERK1/2 MAPK are required for TGF-β1 synthesis induced by activating anti-CD36 mIgA. A, RAWTβRII cells were stimulated with activating anti-CD36 mIgA (2 µg/ml) for the times indicated. Total cell lysates were immunoblotted for phospho-Lyn kinase and the band density was normalized to total Lyn kinase. B, RAWTβRII cells were pretreated with the src-family kinase inhibitor PP2 (0.001 to 100 µM) for 2 h and then stimulated with anti-CD36 mIgA (2 µg/ml). After 6 h, TGF-β1 mRNA expression was analyzed by real-time PCR and normalized to GAPDH. Total TGF-β1 in the conditioned medium was analyzed by ELISA after 18 h. C, A time course of ERK1/2 phosphorylation was assessed by Western blotting in RAWTβRII cells treated with anti-CD36 mIgA (2 µg/ml). Phospho-ERK1/2 band density was normalized to total ERK1/2. D, RAWTβRII cells were preincubated with MEK kinase inhibitor U0126 or inactive analogue U0124 for 2 h and then stimulated with anti-CD36 mIgA for 6 h to detect TGF-β1 mRNA expression or for 18 h to detect secreted TGF-β1 protein as in Figure 1. Values represent means ± SD of six separate experiments.

    Journal: PLoS ONE

    Article Title: Induction of TGF-?1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

    doi: 10.1371/journal.pone.0072772

    Figure Lengend Snippet: Both Lyn kinase and ERK1/2 MAPK are required for TGF-β1 synthesis induced by activating anti-CD36 mIgA. A, RAWTβRII cells were stimulated with activating anti-CD36 mIgA (2 µg/ml) for the times indicated. Total cell lysates were immunoblotted for phospho-Lyn kinase and the band density was normalized to total Lyn kinase. B, RAWTβRII cells were pretreated with the src-family kinase inhibitor PP2 (0.001 to 100 µM) for 2 h and then stimulated with anti-CD36 mIgA (2 µg/ml). After 6 h, TGF-β1 mRNA expression was analyzed by real-time PCR and normalized to GAPDH. Total TGF-β1 in the conditioned medium was analyzed by ELISA after 18 h. C, A time course of ERK1/2 phosphorylation was assessed by Western blotting in RAWTβRII cells treated with anti-CD36 mIgA (2 µg/ml). Phospho-ERK1/2 band density was normalized to total ERK1/2. D, RAWTβRII cells were preincubated with MEK kinase inhibitor U0126 or inactive analogue U0124 for 2 h and then stimulated with anti-CD36 mIgA for 6 h to detect TGF-β1 mRNA expression or for 18 h to detect secreted TGF-β1 protein as in Figure 1. Values represent means ± SD of six separate experiments.

    Article Snippet: Antibodies and reagents TGF-β1 was from R & D Systems (Minneapolis, MN).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    Apoptotic cells induce TGF-β1 production in RAWTβRII macrophages. A, RAW 264.7 macrophages were stably transfected with truncated TGF-β receptor II (RAWTβRII) or empty vector (RAWV). After 48 h, the cells were incubated with TGF-β1 (25 ng/ml) for 6 h. TGF-β1 mRNA expression which was normalized to GAPDH, was measured by real-time PCR and expressed as fold enhancement. B, Human Jurkat T cells were stimulated with UV-irradiation to induce apoptosis. Surface PS exposure was assessed by annexin V staining and cell permeability with propidium iodide as analyzed by flow cytometry (shown as a representative dot plot from five independent experiments). C, Apoptotic or viable Jurkat cells were pretreated with annexin V for 45 min, and incubated with RAWTβRII macrophages for 6 h to measure TGF-β1 mRNA expression. D, Total TGF-β1 in the conditioned medium was analyzed by ELISA after 18 h of co-culture. Values represent means ± SD of five separate experiments. ***, P

    Journal: PLoS ONE

    Article Title: Induction of TGF-?1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

    doi: 10.1371/journal.pone.0072772

    Figure Lengend Snippet: Apoptotic cells induce TGF-β1 production in RAWTβRII macrophages. A, RAW 264.7 macrophages were stably transfected with truncated TGF-β receptor II (RAWTβRII) or empty vector (RAWV). After 48 h, the cells were incubated with TGF-β1 (25 ng/ml) for 6 h. TGF-β1 mRNA expression which was normalized to GAPDH, was measured by real-time PCR and expressed as fold enhancement. B, Human Jurkat T cells were stimulated with UV-irradiation to induce apoptosis. Surface PS exposure was assessed by annexin V staining and cell permeability with propidium iodide as analyzed by flow cytometry (shown as a representative dot plot from five independent experiments). C, Apoptotic or viable Jurkat cells were pretreated with annexin V for 45 min, and incubated with RAWTβRII macrophages for 6 h to measure TGF-β1 mRNA expression. D, Total TGF-β1 in the conditioned medium was analyzed by ELISA after 18 h of co-culture. Values represent means ± SD of five separate experiments. ***, P

    Article Snippet: Antibodies and reagents TGF-β1 was from R & D Systems (Minneapolis, MN).

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, Incubation, Expressing, Real-time Polymerase Chain Reaction, Irradiation, Staining, Permeability, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

    Lyn kinase and ERK1/2 MAPK act by separate pathways for activating anti-CD36 mIgA-induced TGF-β1 synthesis. A, RAWTβRII cells were pretreated with inhibitor PP2 (30 µM) for 2 h prior to stimulation with anti-CD36 mIgA (2 µg/ml) for 60 min. Phosphorylation of Lyn and ERK1/2 were analyzed by Western blotting using total cell lysates. B, RAWTβRII cells were pretreated with inhibitor U0126 or analogue U0124 (1.0 µM) for 2 h and then incubated with anti-CD36 mIgA (2 µg/ml) for 90 min. Total cell lysates were used to analyze phosphorylation of Lyn and ERK1/2. Relative values for phosphorylated kinase versus total kinase were determined by densitometry and expressed as means ± SD of five separate experiments. *** P

    Journal: PLoS ONE

    Article Title: Induction of TGF-?1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

    doi: 10.1371/journal.pone.0072772

    Figure Lengend Snippet: Lyn kinase and ERK1/2 MAPK act by separate pathways for activating anti-CD36 mIgA-induced TGF-β1 synthesis. A, RAWTβRII cells were pretreated with inhibitor PP2 (30 µM) for 2 h prior to stimulation with anti-CD36 mIgA (2 µg/ml) for 60 min. Phosphorylation of Lyn and ERK1/2 were analyzed by Western blotting using total cell lysates. B, RAWTβRII cells were pretreated with inhibitor U0126 or analogue U0124 (1.0 µM) for 2 h and then incubated with anti-CD36 mIgA (2 µg/ml) for 90 min. Total cell lysates were used to analyze phosphorylation of Lyn and ERK1/2. Relative values for phosphorylated kinase versus total kinase were determined by densitometry and expressed as means ± SD of five separate experiments. *** P

    Article Snippet: Antibodies and reagents TGF-β1 was from R & D Systems (Minneapolis, MN).

    Techniques: Activated Clotting Time Assay, Western Blot, Incubation

    Lyn kinase and ERK1/2 are involved in CD36 mediated TGF-β1 induction by apoptotic cells. A, RAWTβRII cells were cultured in the presence of viable or apoptotic Jurkat cells for the time indicated. Total cell lysates were used for analyzing phosphorylation of ERK1/2 MAPK and Lyn kinase by Western blotting. B, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h were incubated with apoptotic Jurkat cells for 60 or 90 min to analyze phosphorylation of Lyn kinase or ERK1/2, respectively. C, RAWTβRII cells were preincubated with PP2 (30 µM) or U0126 (1 µM) for 2h and then co-cultured with apoptotic Jurkat cells. TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. Values represent means ± SD of six separate experiments. ***, P

    Journal: PLoS ONE

    Article Title: Induction of TGF-?1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

    doi: 10.1371/journal.pone.0072772

    Figure Lengend Snippet: Lyn kinase and ERK1/2 are involved in CD36 mediated TGF-β1 induction by apoptotic cells. A, RAWTβRII cells were cultured in the presence of viable or apoptotic Jurkat cells for the time indicated. Total cell lysates were used for analyzing phosphorylation of ERK1/2 MAPK and Lyn kinase by Western blotting. B, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h were incubated with apoptotic Jurkat cells for 60 or 90 min to analyze phosphorylation of Lyn kinase or ERK1/2, respectively. C, RAWTβRII cells were preincubated with PP2 (30 µM) or U0126 (1 µM) for 2h and then co-cultured with apoptotic Jurkat cells. TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. Values represent means ± SD of six separate experiments. ***, P

    Article Snippet: Antibodies and reagents TGF-β1 was from R & D Systems (Minneapolis, MN).

    Techniques: Cell Culture, Western Blot, Transfection, Incubation, Expressing

    Stimulation with activating anti-CD36 mIgA induces TGF-β1 synthesis. A, RAWTβRII cells were cultured with activating anti-CD36 (JC63.1) mIgA or isotype control (2 µg/ml) for the times indicated. TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. B, RAWTβRII cells were pre-treated with the indicated concentrations of actinomycin D or cycloheximide for 1 h, before stimulation for 6 h and TGF-β1 mRNA expression was analyzed as above. C, RAWTβRII cells were cultured in the presence of PMA (50 nM) for 18 h to increase the steady state TGF-β1 mRNA level, and then the cells were incubated with actinomycin D (10 µg/ml) in the presence or absence of anti-CD36 mIgA (2 µg/ml) for the times indicated. D, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h were incubated with anti-CD36 mIgA (2 µg/ml) or isotype control (2 µg/ml) for 6 h or 18 h to analyze TGF-β1 mRNA expression or secreted total TGF-β1 protein respectively, as in Figure 1. Values represent means ± SD of five separate experiments. **, P

    Journal: PLoS ONE

    Article Title: Induction of TGF-?1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

    doi: 10.1371/journal.pone.0072772

    Figure Lengend Snippet: Stimulation with activating anti-CD36 mIgA induces TGF-β1 synthesis. A, RAWTβRII cells were cultured with activating anti-CD36 (JC63.1) mIgA or isotype control (2 µg/ml) for the times indicated. TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. B, RAWTβRII cells were pre-treated with the indicated concentrations of actinomycin D or cycloheximide for 1 h, before stimulation for 6 h and TGF-β1 mRNA expression was analyzed as above. C, RAWTβRII cells were cultured in the presence of PMA (50 nM) for 18 h to increase the steady state TGF-β1 mRNA level, and then the cells were incubated with actinomycin D (10 µg/ml) in the presence or absence of anti-CD36 mIgA (2 µg/ml) for the times indicated. D, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h were incubated with anti-CD36 mIgA (2 µg/ml) or isotype control (2 µg/ml) for 6 h or 18 h to analyze TGF-β1 mRNA expression or secreted total TGF-β1 protein respectively, as in Figure 1. Values represent means ± SD of five separate experiments. **, P

    Article Snippet: Antibodies and reagents TGF-β1 was from R & D Systems (Minneapolis, MN).

    Techniques: Cell Culture, Expressing, Incubation, Transfection

    The transforming growth factor (TGF)-β pathway is activated during culture of mouse ATII cells. ATII cells were freshly isolated (D0) or cultured for 7 days on tissue culture plastic. The mRNA expression levels of Tgf-β1 , Tgf-β2

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Transdifferentiation of alveolar epithelial type II to type I cells is controlled by opposing TGF-β and BMP signaling

    doi: 10.1152/ajplung.00032.2013

    Figure Lengend Snippet: The transforming growth factor (TGF)-β pathway is activated during culture of mouse ATII cells. ATII cells were freshly isolated (D0) or cultured for 7 days on tissue culture plastic. The mRNA expression levels of Tgf-β1 , Tgf-β2

    Article Snippet: Recombinant human TGF-β1 and BMP-4 were obtained from R & D Systems (Minneapolis, MN).

    Techniques: Isolation, Cell Culture, Expressing

    TGF-β1 treatment promotes ATII transdifferentiation mainly by promoting loss of ATII phenotype. ATII cells were cultured in the absence or presence of human (h) TGF-β1 (4 ng/ml) through 7 days. A : the mRNA expression of Sftpa , Sftpb , and

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Transdifferentiation of alveolar epithelial type II to type I cells is controlled by opposing TGF-β and BMP signaling

    doi: 10.1152/ajplung.00032.2013

    Figure Lengend Snippet: TGF-β1 treatment promotes ATII transdifferentiation mainly by promoting loss of ATII phenotype. ATII cells were cultured in the absence or presence of human (h) TGF-β1 (4 ng/ml) through 7 days. A : the mRNA expression of Sftpa , Sftpb , and

    Article Snippet: Recombinant human TGF-β1 and BMP-4 were obtained from R & D Systems (Minneapolis, MN).

    Techniques: Cell Culture, Expressing

    TGF-β and BMP signaling antagonizes each other during culture. Primary ATII cells were treated with hTGF-β1 (4 ng/ml) ( A ) or hBMP-4 (200 ng/ml) ( B ). Cells treated with TGF-β1 were harvested on days 1 and 3 , whereas cells treated

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Transdifferentiation of alveolar epithelial type II to type I cells is controlled by opposing TGF-β and BMP signaling

    doi: 10.1152/ajplung.00032.2013

    Figure Lengend Snippet: TGF-β and BMP signaling antagonizes each other during culture. Primary ATII cells were treated with hTGF-β1 (4 ng/ml) ( A ) or hBMP-4 (200 ng/ml) ( B ). Cells treated with TGF-β1 were harvested on days 1 and 3 , whereas cells treated

    Article Snippet: Recombinant human TGF-β1 and BMP-4 were obtained from R & D Systems (Minneapolis, MN).

    Techniques:

    Vitamin D hydroxyderivatives down-regulate mRNAs of types I and III collagens and hyaluronan synthase 2 in TGFβ1-stimulated fibroblasts. Expression of COL1A2 mRNA in line Fib1 treated with 10 −10 M vitamin D hydroxy-derivatives (A); COL3A1 mRNA in line Fib1 treated with 10 −8 M vitamin D hydroxy-derivatives (B); and HAS2 in line Z4 treated with 10 −8 M vitamin D hydroxyderivatives (C) are shown. Results are expressed as mean ± SD, with P values of EtOH + TGF-β1 vs different treatments as indicated above connecting lines.

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: 20S-Hydroxyvitamin D3, Noncalcemic Product of CYP11A1 Action on Vitamin D3, Exhibits Potent Antifibrogenic Activity in Vivo

    doi: 10.1210/jc.2012-3074

    Figure Lengend Snippet: Vitamin D hydroxyderivatives down-regulate mRNAs of types I and III collagens and hyaluronan synthase 2 in TGFβ1-stimulated fibroblasts. Expression of COL1A2 mRNA in line Fib1 treated with 10 −10 M vitamin D hydroxy-derivatives (A); COL3A1 mRNA in line Fib1 treated with 10 −8 M vitamin D hydroxy-derivatives (B); and HAS2 in line Z4 treated with 10 −8 M vitamin D hydroxyderivatives (C) are shown. Results are expressed as mean ± SD, with P values of EtOH + TGF-β1 vs different treatments as indicated above connecting lines.

    Article Snippet: The confluent fibroblasts were preincubated in serum-free media for 24 hours, and vitamin D hydroxyderivatives (10−9 or 10−10 M) were added to 3 replicate wells 2 hours prior to addition of recombinant human TGF-β1 (R & D Systems, Minneapolis, Minnesota) to a final concentration of 5 ng/mL.

    Techniques: Expressing

    Vitamin D hydroxyderivatives inhibit total collagen and hyaluronan production stimulated by TGF-β1. Scleroderma human dermal fibroblast line 08 (A and B) or normal human dermal fibroblasts line 442 (C) were exposed to 20(OH)D 3 , 20,23(OH) 2 D 3 , or 1,25(OH) 2 D 3 and then treated with TGF-β1 as described. Total collagen (A and C) and hyaluronan (A) and collagen type 1 (B) were measured versus PBS + EtOH and TGF-β1 + EtOH controls, respectively. Data are presented as mean ± SEM of 3 replicates, and P values for statistically significant differences between control and treatments are shown above the bars. Results were confirmed using 2 additional normal human dermal fibroblast lines (data not shown).

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: 20S-Hydroxyvitamin D3, Noncalcemic Product of CYP11A1 Action on Vitamin D3, Exhibits Potent Antifibrogenic Activity in Vivo

    doi: 10.1210/jc.2012-3074

    Figure Lengend Snippet: Vitamin D hydroxyderivatives inhibit total collagen and hyaluronan production stimulated by TGF-β1. Scleroderma human dermal fibroblast line 08 (A and B) or normal human dermal fibroblasts line 442 (C) were exposed to 20(OH)D 3 , 20,23(OH) 2 D 3 , or 1,25(OH) 2 D 3 and then treated with TGF-β1 as described. Total collagen (A and C) and hyaluronan (A) and collagen type 1 (B) were measured versus PBS + EtOH and TGF-β1 + EtOH controls, respectively. Data are presented as mean ± SEM of 3 replicates, and P values for statistically significant differences between control and treatments are shown above the bars. Results were confirmed using 2 additional normal human dermal fibroblast lines (data not shown).

    Article Snippet: The confluent fibroblasts were preincubated in serum-free media for 24 hours, and vitamin D hydroxyderivatives (10−9 or 10−10 M) were added to 3 replicate wells 2 hours prior to addition of recombinant human TGF-β1 (R & D Systems, Minneapolis, Minnesota) to a final concentration of 5 ng/mL.

    Techniques: