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  • 83
    IQ Products anti transforming growth factor β tgf β
    Anti Transforming Growth Factor β Tgf β, supplied by IQ Products, used in various techniques. Bioz Stars score: 83/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems anti transforming growth factor β tgf β
    Anti Transforming Growth Factor β Tgf β, supplied by R&D Systems, used in various techniques. Bioz Stars score: 76/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc tgfβ
    Western-blot analysis of AG1 and <t>TGFβ</t> protein levels Western blottings were performed using whole-brain lysates and the relative band density of each antigen compared with β-actin was calculated for AG1 and for TGFβ. All band intensities were measured using the FluorChem Q (Cell Biosciences). Lanes 1–3, WT; lanes 4–6, APPSw; lanes 7–9, APPSw/NOS2 −/− ; * P
    Tgfβ, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc tgfβ antibodies
    Effect of TAZ or YAP knockdown plus and minus <t>TGFβ</t> on transwell migration of canine OSA cell lines. Representative images of migrated cells stained with 0.1% crystal violet taken with 10X objective for primary cell lines ( a ) and metastatic cell lines ( b ). Graphs depict the absorbance values of migrated of cells, as determined by extraction of crystal violet dye from transwells using 10% acetic acid and spectrophotometer reading at 590 nm. Readings were first blank-corrected to insert containing no cells. Error bars depict average ± SEM from at least three independent experiments plated in duplicate. Asterisks depict statistical differences as determined by a two-way ANOVA with post-hoc Tukey-Kramer, * indicates p
    Tgfβ Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tgf β
    Cytokine concentrations in one-way MLR supernatants according to flow cytometric bead arrays ((a) IFN- γ ; (b) TNF- α ; (c) IL-10; (d) IL-4) and ELISA ((e) <t>TGF-</t> β ) presented as scatter plots with mean bar. “∗” P
    Tgf β, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3054 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson tgf β
    Coculture of CD4 + CD25 + and CD4 + CD25 − T cells results in high level IL-10 production. CD4 + CD25 + and CD4 + CD25 − T cells were MACS ® sorted from PBMCs of healthy individuals. These cells were either cultured alone or at a 1:1 ratio and activated with platebound anti-CD3 and soluble anti-CD28 (10 μg/ml, respectively). (A) After various time points supernatants were analyzed for cytokine production by ELISA. IL-10 production peaked 48 h after onset of culture and was markedly higher in the coculture of CD4 + CD25 + and CD4 + CD25 − T cells than in the cultures of each of the cell types alone. A representative out of five independent standardized experiments is shown. No elevated levels of INF-α or <t>TGF-β</t> could be measured (data not shown). (B) The different T cell populations were also activated with mature allogeneic DCs (DC/T cell ratio 1:20) compared with anti-CD3 and anti-CD28 (10 μg/ml, respectively). Cytokines were measured 48 h after onset of culture. Results were similar in five independent experiments. (C) For the last 6 h of activation with anti-CD3 and anti-CD28 2 μM monensin was added to the cultures. Staining of CD4 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for intracellular IL-10 using PE-conjugated specific Abs. One of five independent experiments is shown.
    Tgf β, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 797 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend tgf β
    Th17 cells expressing RANKL promote osteoclast maturation A. Naïve CD4+ T cells were differentiated into Th17 cells for 5 days and the surface expression of RANKL and CD4 and the intracellular expression of IL-17 were detected by flow cytometry. B. The osteoclast precursor cell line RAW 264.7 was cultured with or without RANKL (50 ng/ml) and SDF-1a (20ng/ml) for 3 days. Naïve CD4+ T cells were added to the cultures with or without Th17-skewing cytokines (IL-6, IL-23, and <t>TGF-β).</t> After 3 days, RAW 264.7 cells were fixed and stained for TRAP activity. Total TRAP-positive cells in the absence of RANKL and SDF-1α C . TRAP-positive multinucleated cells in the presence of RANKL and SDF-1α were counted. D . Data are presented as means ± SEM (* P
    Tgf β, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology tgf β
    miRNA-30e regulates <t>Snai1/TGF-β/Nox4</t> protein expression. (A) Snai1, (B) TGF-β, (C) Smad2 and (D) Nox4 protein expression by statistical analysis, and (E) western blotting analysis. ## P
    Tgf β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems tgf β
    Functional characterization of Treg after EP treatment. (A) CTLA-4 protein expression in CD25 + cells purified from the pool of lymphoid tissues, normalized to the expression of β-actin, along with the representative blot. (B) The proportion of <t>TGF-β-expressing</t> cells within Treg, along with representative dot plots. (C) IL-10 protein expression in CD25 + cells purified from a pool of lymphoid tissues, normalized to the expression of β-actin, along with the representative blot. (D) The proportion of IL-10-expressing Treg within pancreatic infiltrates, along with representative dot plots. (E) The level of inhibition of effector T cell (Teff–CD4 + CD25 − ) proliferation after co-culture with Treg (CD4 + CD25 + ) cells purified from a pool of lymphoid tissues of MLDS or MLDS+EP-treated mice. Proliferation was measured after 72 h of incubation by CFSE dilution (in Teff). (F) Proportion of divided Teff, or Teff cultured in the presence of Treg (1:1 ratio). Representative dot plots are given below. All measurements were performed on samples from 7 animals per group. * p
    Tgf β, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 5042 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam tgf β
    Expression of fibronectin in cells, tissues and tumours. ( a ) The morphology of 4T1 cells with and without <t>TGF-β</t> induction (15 ng ml −1 , 5 days). Images were taken by phase-contrast microscopy; all scale bars, 50 μm. ( b ) RT–PCR analysis demonstrates that treatment with TGF-β induces upregulation of fibronectin and downregulation of E-cadherin, which are characteristic features of EMT, data represent the mean±s.d., n =3. ( c ) Representative western blots showing fibronectin expression in normal tissues, and in primary and metastatic 4T1 breast tumours in Balb/c mice. ( d ) Densitometric analysis of the expression of fibronectin normalized to that of β-actin. Values represent mean±s.d., n =3.
    Tgf β, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tgf β
    Representative image of morphological changes in HPMCs. (Upper left) HPMCs cultured in control medium. (Upper middle) HPMCs cultured in medium containing 100 μg/ml of PSK. (Upper right) HPMCs cultured in medium containing 500 μg/ml of PSK. (Lower left) HPMCs cultured in medium containing 10 ng/ml of <t>TGF-β.</t> (Lower middle) HPMCs cultured in 10 ng/ml of TGF-β and 100 μg/ml of PSK. (Lower right) HPMCs cultured in 10 ng/ml of TGF-β and 500 μg/ml of PSK. HPMCs cultured in each condition for 72 h were visualized by phase contrast microscopy at magnification, ×200. HPMCs, human peritoneal mesothelial cells; PSK, protein-bound polysaccharide K.
    Tgf β, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM tgf β
    Induction of TAMs and CAFs by IL-6 in human bladder cancer tissues and confirmation by in vitro experiments. (A) Representative expression status for IL-6 in human bladder cancer tissues. Images were captured at 200× magnification. (B) The interrelationship between the percentage of IL-6-positive cancer cells and the number of tumor-infiltrating TAMs was examined using Spearman’s correlation. Spearman r was found to be 0.68 (95% confidence interval, 058–0.76). (C) Comparison of the percentage of IL-6-positive cancer with the induction level of CAFs (CAF score). (D) Morphological changes of THP-1 cells by PMA and IL-4 treatment. THP-1 cells were treated with 200 nM PMA for 24 h to differentiate them into resting macrophages (middle), followed by treatment with IL-4 (20 ng/mL) for 48 h (right). (E) Western blot analysis for confirming the generation of TAMs and CAFs. THP-1 cells were treated with a combination of PMA and 20 ng/mL of CXCL1, IL-6, or <t>TGF-β,</t> separately. Upregulation of CD204 indicates differentiation into TAMs. NIH3T3 cells were treated with 20 ng/mL of CXCL1, IL-6, or TGF-β, separately. Upregulation of αSMA indicates the activation of fibroblasts, which are known to be CAFs.
    Tgf β, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche tgf β
    MG induces YAP co-transcriptional activity in breast cancer cells. ( A ) Western blot detection of YAP in MDA-MB-231 cells under the indication conditions. Immunoblot is representative of three independent experiments. ( B ) Western blot of Phospho-Smad2/3 and Smad2/3 in MDA-MB-231 treated with MG until confluence and then with <t>TGF-β</t> during 2 hr. β-actin is used for normalization. ( C and D ) YAP mRNA and protein level assessed by qRT-PCR and Western blot, respectively, in MDA-MB-231 cells silenced for YAP (siYAP#1 and #2) and treated in the same conditions as in Figure 5D . Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. ( E ) YAP (Cell Signaling, 4912) and TEAD1 IF co-localization in MDA-MB-231 cells cultured under low (Sparse) density used as positive control and in high-density cultured cells (Confluent) in presence of MG. Magnification 630x. Data are representative of two independent experiments. ( F ) Proliferation assay on GLO1 -depleted MDA-MB-231 (sh GLO1 #1 and #2) silenced or not for YAP (siYAP#1 and #2) at different time points. Data (72 hr) were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. All immunoblots are representative of three independent experiments. ***p
    Tgf β, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    USCN Life tgf β
    The serum cytokine levels of <t>TGF-β,</t> IL-6 and IL-23 was measured by ELISA in MS patients and healthy controls. Comparison of TGF-β (A), IL-6 (B), IL-23 (C) cytokines concentrations between HC, RRMS patients in relapse and remission phase, SPMS and PPMS patients. Pearson’s correlation and regression tests between TGF-β (D), IL-6 (E), IL-23 (F) and Tc17 cells in MS patients. One-way ANOVA was used to test for differences between the groups. Subsequent multiple comparison pairwise test was Bonferroni. * P-value
    Tgf β, supplied by USCN Life, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare tgf β
    qRT-PCR analysis of selected genes in DLE lesional and normal skin. a – k RNA expression of general macrophage markers (CD163 and CD68) ( a , b ), M1 macrophage-related markers (CD127, TNF-α, CXCL10, STAT1, and CCL5) ( c – g ), and M2 macrophage-related markers <t>(TGF-β,</t> CD206, CD209, and arginase-1) ( h – k ) was compared in DLE lesional (n = 17) and normal (n = 12) skin via qRT-PCR. Multiple general and M1 macrophage-related markers were upregulated in DLE lesional skin. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. CCL chemokine (C-C motif) ligand, CXCL chemokine (C-X-C motif) ligand, DLE discoid lupus erythematosus, qRT-PCR quantitative real-time polymerase chain reaction, STAT signal transducer and activator of transcription, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha
    Tgf β, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 97/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra tgf β
    Mast cell expression of IL-6 in parenchyma in patients with CF (A), and mast cell expression of <t>TGF-β</t> in parenchyma in IPF patients (B) . Overall significance for total mast cell expression in different parenchyma is shown in each picture. Significance to healthy controls is shown as: p
    Tgf β, supplied by Novocastra, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega tgf β
    Identification of endoglin as a direct target of miR‐342‐5p. A, The 3′ UTR of human and mouse endoglin mRNA were aligned with Hsa‐miR‐342‐5p and Mmu‐miR‐342‐5p. Complementary sequences were marked in red. B and C, HUVEC s were transfected with miR‐342‐5p, miR‐342‐5p ASO , or control. The level of miR‐342‐5p was determined with qRT ‐ PCR (B). Endoglin expression was determined by using Western blot (C). Bands were quantitatively compared between groups. D, Total RNA was prepared from HUVEC s transfected with miR‐342‐5p or retina tissues derived from pups at postnatal day 7 that had accepted intravitreal injection of miR‐342‐5p on postnatal day 3. Endoglin mRNA level was determined by using qRT ‐ PCR . E, Reporter assay. HeLa cells were cotransfected with pGL 3‐endoglin wild type or pGL 3‐endoglin mutant, together with increasing amount of miR‐342‐5p. Cells were harvested 24 hours after the transfection, and luciferase activity was analyzed. F, Liver sinusoid endothelial cells were isolated from endothelial‐specific Notch‐activating mice and control mice, and expression of endoglin was determined by using qRT ‐ PCR . G, HUVEC s transfected with miR‐342‐5p or control were cultured in the presence of <t>TGF</t> ‐β for 30 minutes. The phosphorylation of Smad1/5 and Smad2/3 was determined by using Western blot and quantitatively compared. H, HUVEC s were transfected with control, miR‐342‐5p or miR‐342‐5p plus endoglin and stimulated with vascular endothelial growth factor. The total and phosphorylated Akt were determined using Western blot. Bars indicate mean± SD (n=5), * P
    Tgf β, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems panspecific transforming growth factor β tgf β
    Identification of endoglin as a direct target of miR‐342‐5p. A, The 3′ UTR of human and mouse endoglin mRNA were aligned with Hsa‐miR‐342‐5p and Mmu‐miR‐342‐5p. Complementary sequences were marked in red. B and C, HUVEC s were transfected with miR‐342‐5p, miR‐342‐5p ASO , or control. The level of miR‐342‐5p was determined with qRT ‐ PCR (B). Endoglin expression was determined by using Western blot (C). Bands were quantitatively compared between groups. D, Total RNA was prepared from HUVEC s transfected with miR‐342‐5p or retina tissues derived from pups at postnatal day 7 that had accepted intravitreal injection of miR‐342‐5p on postnatal day 3. Endoglin mRNA level was determined by using qRT ‐ PCR . E, Reporter assay. HeLa cells were cotransfected with pGL 3‐endoglin wild type or pGL 3‐endoglin mutant, together with increasing amount of miR‐342‐5p. Cells were harvested 24 hours after the transfection, and luciferase activity was analyzed. F, Liver sinusoid endothelial cells were isolated from endothelial‐specific Notch‐activating mice and control mice, and expression of endoglin was determined by using qRT ‐ PCR . G, HUVEC s transfected with miR‐342‐5p or control were cultured in the presence of <t>TGF</t> ‐β for 30 minutes. The phosphorylation of Smad1/5 and Smad2/3 was determined by using Western blot and quantitatively compared. H, HUVEC s were transfected with control, miR‐342‐5p or miR‐342‐5p plus endoglin and stimulated with vascular endothelial growth factor. The total and phosphorylated Akt were determined using Western blot. Bars indicate mean± SD (n=5), * P
    Panspecific Transforming Growth Factor β Tgf β, supplied by R&D Systems, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems pan specific transforming growth factor β tgf β antibody
    Identification of endoglin as a direct target of miR‐342‐5p. A, The 3′ UTR of human and mouse endoglin mRNA were aligned with Hsa‐miR‐342‐5p and Mmu‐miR‐342‐5p. Complementary sequences were marked in red. B and C, HUVEC s were transfected with miR‐342‐5p, miR‐342‐5p ASO , or control. The level of miR‐342‐5p was determined with qRT ‐ PCR (B). Endoglin expression was determined by using Western blot (C). Bands were quantitatively compared between groups. D, Total RNA was prepared from HUVEC s transfected with miR‐342‐5p or retina tissues derived from pups at postnatal day 7 that had accepted intravitreal injection of miR‐342‐5p on postnatal day 3. Endoglin mRNA level was determined by using qRT ‐ PCR . E, Reporter assay. HeLa cells were cotransfected with pGL 3‐endoglin wild type or pGL 3‐endoglin mutant, together with increasing amount of miR‐342‐5p. Cells were harvested 24 hours after the transfection, and luciferase activity was analyzed. F, Liver sinusoid endothelial cells were isolated from endothelial‐specific Notch‐activating mice and control mice, and expression of endoglin was determined by using qRT ‐ PCR . G, HUVEC s transfected with miR‐342‐5p or control were cultured in the presence of <t>TGF</t> ‐β for 30 minutes. The phosphorylation of Smad1/5 and Smad2/3 was determined by using Western blot and quantitatively compared. H, HUVEC s were transfected with control, miR‐342‐5p or miR‐342‐5p plus endoglin and stimulated with vascular endothelial growth factor. The total and phosphorylated Akt were determined using Western blot. Bars indicate mean± SD (n=5), * P
    Pan Specific Transforming Growth Factor β Tgf β Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IQ Products tgf β
    Oral pretreatment of mice with M. leprae -Hsp65-producing L. lactis was associated with an increased of CD4+LAP+ regulatory T cells expressing <t>TGF-β</t> in mice. C57BL/6 mice were fed or not (Naïve) medium (EAE), control (CT-LL+EAE) or M. leprae -Hsp65-producing L. lactis (Hsp65-LL+EAE) for four days and EAE was induced ten days later. After 1, 2, 4 and 14 days, mice were killed and inguinal (ILN; A) and mesenteric lymph nodes (MLN; B) as well as spleen (SPL; C) removed. Cells were stained with FITC-anti-CD4, PE-anti-CD25, Bio-LAP and CY-STV. Graphs are shown as mean ± SEM. ANOVA, post-test Tukey. *Statistically different from EAE group; p
    Tgf β, supplied by IQ Products, used in various techniques. Bioz Stars score: 96/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genzyme tgf β
    The effects of bradykinin B1 (BKB1) and B2 (BKB2) receptor antagonists on the release of IL-8 ( A ), G-CSF ( B ), MCP-1 ( C ), and <t>TGF-β</t> ( D ) from A549 cell monolayer after a 72-hour incubation ( n = 5). * P
    Tgf β, supplied by Genzyme, used in various techniques. Bioz Stars score: 94/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec tgf β
    Th17 cell ectonucleotidase expression is driven by tumor-derived <t>TGF-β</t> and IL-6. ( A ) mRNA was extracted from 36 breast cancer tumor samples and the expression of IL17, ENTPD1 and NT5E was determined using RT-qPCR. The correlation between IL17
    Tgf β, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen tgf β
    Defective expression and activation of <t>TGF-β</t> in mice infected with MHV68-IκBαM. A: Western blot assay for latent form of TGF-β in BAL collected from naïve (N) and MHV68-MR and MHV68-IκBαM–infected
    Tgf β, supplied by Pharmingen, used in various techniques. Bioz Stars score: 95/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc tgf β
    Composite profile of Western blot analysis for p16 before and after exposure of proliferating KCs to anti-proliferative reagents (TNF-α, IFN-γ, <t>TGF-β,</t> and TPA) for the indicated time intervals ( A ) and after 48 hours including pretreatment with the MEK inhibitor (PD98059) ( C ). B: Total Ras protein level and activity in KCs before and after exposure to TPA or IFN-γ. D: Western blot analysis of KCs before and after suspension in methylcellulose to detect p12 and p16 levels in the absence and presence of the PKC inhibitor, GF.
    Tgf β, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MorphoSys tgf β
    Composite profile of Western blot analysis for p16 before and after exposure of proliferating KCs to anti-proliferative reagents (TNF-α, IFN-γ, <t>TGF-β,</t> and TPA) for the indicated time intervals ( A ) and after 48 hours including pretreatment with the MEK inhibitor (PD98059) ( C ). B: Total Ras protein level and activity in KCs before and after exposure to TPA or IFN-γ. D: Western blot analysis of KCs before and after suspension in methylcellulose to detect p12 and p16 levels in the absence and presence of the PKC inhibitor, GF.
    Tgf β, supplied by MorphoSys, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    4Gene tgf β
    Composite profile of Western blot analysis for p16 before and after exposure of proliferating KCs to anti-proliferative reagents (TNF-α, IFN-γ, <t>TGF-β,</t> and TPA) for the indicated time intervals ( A ) and after 48 hours including pretreatment with the MEK inhibitor (PD98059) ( C ). B: Total Ras protein level and activity in KCs before and after exposure to TPA or IFN-γ. D: Western blot analysis of KCs before and after suspension in methylcellulose to detect p12 and p16 levels in the absence and presence of the PKC inhibitor, GF.
    Tgf β, supplied by 4Gene, used in various techniques. Bioz Stars score: 98/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cambrex tgf β
    Composite profile of Western blot analysis for p16 before and after exposure of proliferating KCs to anti-proliferative reagents (TNF-α, IFN-γ, <t>TGF-β,</t> and TPA) for the indicated time intervals ( A ) and after 48 hours including pretreatment with the MEK inhibitor (PD98059) ( C ). B: Total Ras protein level and activity in KCs before and after exposure to TPA or IFN-γ. D: Western blot analysis of KCs before and after suspension in methylcellulose to detect p12 and p16 levels in the absence and presence of the PKC inhibitor, GF.
    Tgf β, supplied by Cambrex, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tebu-bio sa tgf β
    PAR‐1 activation induces mesenchymal transition of imPTECs. A, Relative mRNA expression levels of AQP‐1, ZO‐1, vimentin, α‐SMA, fibronectin, and collagen I in imPTECs 24 hours after stimulation with thrombin (1 U/mL) or <t>TGF‐Β</t> (5 ng/mL). Indicated is the average of three independent experiments. * P
    Tgf β, supplied by tebu-bio sa, used in various techniques. Bioz Stars score: 96/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Torrey Pines Biolabs tgf β
    Treatment with IL-2C increased the expression of M2-associated markers in the contusion margin. (A) Co-localization of CD206 with the microglia marker Iba1 showed that IL-2C increased the number of M2 microglia on day 3 post-injury in the contusion area. (B) Cell count analyses indicated that IL-2C significantly increased the amount of CD206 and Iba-1-double-positive cells. (C,D) The traumatic brain injury TBI-induced expression of <t>TGF-β</t> in the ipsilateral cortices on days 1 and 3 post-injury was increased by treatment with IL-2C. (E,F) Immunohistochemistry showed that treatment with IL-2C significantly increased the number of arginase-1 + cells around the lesion area. Data are presented as the mean ± SD; * p
    Tgf β, supplied by Torrey Pines Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Elabscience tgf β
    Expression levels of cytokines (A) IL-6, (B) IL-10, (C) IL-23 and (D) <t>TGF-β,</t> as determined by ELISA. The horizontal line represents the mean values for each group. IL, interleukin; TGF, transforming growth factor.
    Tgf β, supplied by Elabscience, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bender MedSystems tgf β
    a Effect of VCN-2 hydrocolloid film on expression of mediators after 14 days treatment was detected by Western blotting. Down-regulation of various anti-inflammatory and up-regulation of expression of several wound healing markers in skin tissues were observed. β-actin acted as control marker. b Effect of VCN-2 film on expression of mediators after 14 days topically treatment was detected by immunoblotting. A graph represented densitometry analysis results of the effect of VCN-2 on proteins expression proinflammatory mediators ( c ) anti-HIF1α, ( d ) MMP-9, and ( e ) VEGF and <t>TGF-β.</t> All data are expressed as mean ± SEM. ### p
    Tgf β, supplied by Bender MedSystems, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nordic BioSite tgf β
    GD2-CAR-engineered T cells, expanded by the allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP), are more resistant to immunosuppressive cytokines and oxidative and apoptotic stress than GD2-CAR T cells expanded by the rapid expansion protocol (REP). ( a ) An illustration of the GD2-CAR-encoding lentiviral vector used to transduce T cells. ( b ) CD8 + T cells were transduced, isolated by magnetic beads, and expanded by the REP or AEP protocols. The starting number of GD2-CAR T cells was 1 × 10 5 for both protocols and a ratio of 1:1:4 (T-cell:mDC:ASAL) was used in the AEP protocol. Fold expansion of GD2-CAR T cells is presented. ( c ) The cytotoxic ability of GD2-CAR T cells was analyzed by coculture with luciferase-expressing GD2 + (IMR-32) or GD2 − (SK-N-FI) neuroblastoma target cells at different E:T ratios. Viability of target cells were measured by luminescence and related to signals from target cells alone. ( d ) GD2-CAR T cells expanded by the REP and AEP protocols were exposed to either 25 μmol/l H 2 O 2 or 0.1 μmol/l doxorubicin for 24 hours. Viability was analyzed by flow cytometry using Annexin-V and PI staining and relative viability was normalized against nontreated GD2-CAR T cells. ( e–f ) The REP- and AEP-expanded GD2-CAR T cells were treated with <t>IL-10/TGF-β</t> for 4 hours or with H 2 O 2 for 24 hours, followed by 4 hours treatment with IL-10/TGF-β and then cocultured with GD2 + target cells (IMR-32) in the presence of IL-10/TGF-β for 24 hours. ( e ) Flow cytometry was used to analyze CD107a expression on GD2-CAR T cells (CD3 + ) after exposure to IL-10/TGF-β or H 2 O 2 /IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. ( f ) ELISA was used to analyze IFN-γ production from GD2-CAR T cells after exposure to IL-10/TGF-β or H 2 O 2 /IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. ( g ) GD2-CAR T cells were treated with H 2 O 2 for 24 hours, followed by 4 hours labeling with CFSE in the presence of IL-10/TGF-β and then cocultured with GD2 + target cells (IMR-32) in the presence of IL-10/TGF-β for 4 days before proliferation analysis by flow cytometry. ( a–g ) The experiments were performed three times with three new and different donors each time. Error bars represent SD, and statistical significance is depicted by symbols (* P
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    Image Search Results


    Western-blot analysis of AG1 and TGFβ protein levels Western blottings were performed using whole-brain lysates and the relative band density of each antigen compared with β-actin was calculated for AG1 and for TGFβ. All band intensities were measured using the FluorChem Q (Cell Biosciences). Lanes 1–3, WT; lanes 4–6, APPSw; lanes 7–9, APPSw/NOS2 −/− ; * P

    Journal: ASN NEURO

    Article Title: Diverse inflammatory responses in transgenic mouse models of Alzheimer's disease and the effect of immunotherapy on these responses

    doi: 10.1042/AN20110018

    Figure Lengend Snippet: Western-blot analysis of AG1 and TGFβ protein levels Western blottings were performed using whole-brain lysates and the relative band density of each antigen compared with β-actin was calculated for AG1 and for TGFβ. All band intensities were measured using the FluorChem Q (Cell Biosciences). Lanes 1–3, WT; lanes 4–6, APPSw; lanes 7–9, APPSw/NOS2 −/− ; * P

    Article Snippet: The gel was transferred on to a PVDF membrane, and Western-blot analyses were performed for AG1 [Santa Cruz Biotechnology (catalogue no. sc-20150); 1:1000, stains multiple bands in the 37–45 kDa range], TGFβ [Cell Signaling Technology (catalogue no. 3711); 1:1000, stains multiple bands in the 25–45 kDa range] or β-actin [Santa Cruz Biotechnology (catalogue no. sc-1616); 1:1000].

    Techniques: Western Blot

    Effect of LC on Lactate-4.25% dialysate-induced overexpression of CD206, TGF-β, Arg-1 and Ym-1. Values were expressed as the mean ± SD. ( A ) The overexpression of CD206 and TGF-β induced by Lactate-4.25% dialysate was evidently downregulated by LC treatment measured by Western blotting; ( B ) The relative protein level of CD206 normalized by GAPDH; ( C ) The relative protein level of TGF-β normalized by GAPDH; ( D ) The mRNA level of Arg-1 and Ym-1 were depressed by LC treatment. # p

    Journal: International Journal of Molecular Sciences

    Article Title: The Role of Peritoneal Alternatively Activated Macrophages in the Process of Peritoneal Fibrosis Related to Peritoneal Dialysis

    doi: 10.3390/ijms140510369

    Figure Lengend Snippet: Effect of LC on Lactate-4.25% dialysate-induced overexpression of CD206, TGF-β, Arg-1 and Ym-1. Values were expressed as the mean ± SD. ( A ) The overexpression of CD206 and TGF-β induced by Lactate-4.25% dialysate was evidently downregulated by LC treatment measured by Western blotting; ( B ) The relative protein level of CD206 normalized by GAPDH; ( C ) The relative protein level of TGF-β normalized by GAPDH; ( D ) The mRNA level of Arg-1 and Ym-1 were depressed by LC treatment. # p

    Article Snippet: Immunofluorescence Paraffin-embedded visceral peritoneum sections (6 μm) were used to examine expression of TGF-β (CST, Boston, MA, USA) secreted by M2, surface markers of macrophages, such as F4/80 (Abcam, Cambridge, UK), CD206 (Abcam, Cambridge, UK) and CCR7 (Abcam, Cambridge, UK), and the degree of fibrosis with type I collagen (Col-I, Southern Biotech, Birmingham, AL, USA) and fibronectin (NOVUS, Littleton, CO, USA).

    Techniques: Over Expression, Western Blot

    Liposome-encapsulated clodronate (LC) depleted almost all the M2 in the peritoneum without an effect on M1. ( A ) The number of M1 didn’t change among the four groups. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CCR7; co-location signals were identified as M1; ( B ) The number of M2 significantly increased after injecting Lactate-4.25% dialysate; liposome-encapsulated PBS (LP) didn’t affect M2 infiltration, but LC depleted peritoneal M2 efficiently. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CD206; co-location signals were identified as M2; ( C ) TGF-β was expressed by M2, and LC inhibited the expression of TGF-β by depletion of M2. Blue corresponds to nuclear staining, green corresponds to F4/80, red corresponds to CD206 and yellow corresponds to TGF-β staining.

    Journal: International Journal of Molecular Sciences

    Article Title: The Role of Peritoneal Alternatively Activated Macrophages in the Process of Peritoneal Fibrosis Related to Peritoneal Dialysis

    doi: 10.3390/ijms140510369

    Figure Lengend Snippet: Liposome-encapsulated clodronate (LC) depleted almost all the M2 in the peritoneum without an effect on M1. ( A ) The number of M1 didn’t change among the four groups. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CCR7; co-location signals were identified as M1; ( B ) The number of M2 significantly increased after injecting Lactate-4.25% dialysate; liposome-encapsulated PBS (LP) didn’t affect M2 infiltration, but LC depleted peritoneal M2 efficiently. Blue corresponds to nuclear staining, green corresponds to F4/80 and red corresponds to CD206; co-location signals were identified as M2; ( C ) TGF-β was expressed by M2, and LC inhibited the expression of TGF-β by depletion of M2. Blue corresponds to nuclear staining, green corresponds to F4/80, red corresponds to CD206 and yellow corresponds to TGF-β staining.

    Article Snippet: Immunofluorescence Paraffin-embedded visceral peritoneum sections (6 μm) were used to examine expression of TGF-β (CST, Boston, MA, USA) secreted by M2, surface markers of macrophages, such as F4/80 (Abcam, Cambridge, UK), CD206 (Abcam, Cambridge, UK) and CCR7 (Abcam, Cambridge, UK), and the degree of fibrosis with type I collagen (Col-I, Southern Biotech, Birmingham, AL, USA) and fibronectin (NOVUS, Littleton, CO, USA).

    Techniques: Staining, Expressing

    Effect of TAZ or YAP knockdown plus and minus TGFβ on transwell migration of canine OSA cell lines. Representative images of migrated cells stained with 0.1% crystal violet taken with 10X objective for primary cell lines ( a ) and metastatic cell lines ( b ). Graphs depict the absorbance values of migrated of cells, as determined by extraction of crystal violet dye from transwells using 10% acetic acid and spectrophotometer reading at 590 nm. Readings were first blank-corrected to insert containing no cells. Error bars depict average ± SEM from at least three independent experiments plated in duplicate. Asterisks depict statistical differences as determined by a two-way ANOVA with post-hoc Tukey-Kramer, * indicates p

    Journal: BMC Veterinary Research

    Article Title: An evaluation of TAZ and YAP crosstalk with TGFβ signalling in canine osteosarcoma suggests involvement of hippo signalling in disease progression

    doi: 10.1186/s12917-018-1651-5

    Figure Lengend Snippet: Effect of TAZ or YAP knockdown plus and minus TGFβ on transwell migration of canine OSA cell lines. Representative images of migrated cells stained with 0.1% crystal violet taken with 10X objective for primary cell lines ( a ) and metastatic cell lines ( b ). Graphs depict the absorbance values of migrated of cells, as determined by extraction of crystal violet dye from transwells using 10% acetic acid and spectrophotometer reading at 590 nm. Readings were first blank-corrected to insert containing no cells. Error bars depict average ± SEM from at least three independent experiments plated in duplicate. Asterisks depict statistical differences as determined by a two-way ANOVA with post-hoc Tukey-Kramer, * indicates p

    Article Snippet: Antibodies and TGFβ Antibodies used in this study included: YAP (#14074), β actin (#4967S), pSmad2 (#3108), and Smad2 (#5339) or Smad2/3 (#5678) for pSmad2 normalization (all from Cell Signaling Technology, New England Biolabs, Ltd., Whitby, ON, Canada).

    Techniques: Migration, Staining, Spectrophotometry

    Effect of TGFβ receptor I inhibition by LY2157299 on TAZ levels and cell proliferation in D17 cells. Representative immuoblots and densitometry analysis of TAZ and pSmad3 protein levels when TAZ levels were depleted (siTAZ) or not (siCtrl), and cells were treated with nothing (Control), 5 ng/mL TGFβ (+ TGFβ), 10 μM of LY2152799 (+ LY) or combination treatment (+ TGFβ + LY). Experimental groups were normalized to loading control β-actin. Graphs depict the average fold change in TAZ or pSmad3 expression relative to siCtrl non-treated group ± SEM from two independent experiments ( a ). LY inhibited Smad3 phosphorylation and decreased TAZ levels compared to non-LY treated cells, suggesting that suppressing active TGFβ signaling could decrease TAZ levels in D17 cells. b Representative immunofluorescence images of the mitotic marker phospho-histone H3 (pHH3) taken at 20X objective lens and graph depicting the relative percentage of pHH3 positive cells; bars depict average ± SEM from three independent experiments. Scale bar = 100 μm

    Journal: BMC Veterinary Research

    Article Title: An evaluation of TAZ and YAP crosstalk with TGFβ signalling in canine osteosarcoma suggests involvement of hippo signalling in disease progression

    doi: 10.1186/s12917-018-1651-5

    Figure Lengend Snippet: Effect of TGFβ receptor I inhibition by LY2157299 on TAZ levels and cell proliferation in D17 cells. Representative immuoblots and densitometry analysis of TAZ and pSmad3 protein levels when TAZ levels were depleted (siTAZ) or not (siCtrl), and cells were treated with nothing (Control), 5 ng/mL TGFβ (+ TGFβ), 10 μM of LY2152799 (+ LY) or combination treatment (+ TGFβ + LY). Experimental groups were normalized to loading control β-actin. Graphs depict the average fold change in TAZ or pSmad3 expression relative to siCtrl non-treated group ± SEM from two independent experiments ( a ). LY inhibited Smad3 phosphorylation and decreased TAZ levels compared to non-LY treated cells, suggesting that suppressing active TGFβ signaling could decrease TAZ levels in D17 cells. b Representative immunofluorescence images of the mitotic marker phospho-histone H3 (pHH3) taken at 20X objective lens and graph depicting the relative percentage of pHH3 positive cells; bars depict average ± SEM from three independent experiments. Scale bar = 100 μm

    Article Snippet: Antibodies and TGFβ Antibodies used in this study included: YAP (#14074), β actin (#4967S), pSmad2 (#3108), and Smad2 (#5339) or Smad2/3 (#5678) for pSmad2 normalization (all from Cell Signaling Technology, New England Biolabs, Ltd., Whitby, ON, Canada).

    Techniques: Inhibition, Expressing, Immunofluorescence, Marker

    Effect of TAZ or YAP knockdown plus and minus TGFβ on cell viability of canine OSA cell lines. Graphs depict the fluorescence readings at 530/590 nm when TAZ ( a ) or YAP ( b ) was depleted in OVC-cOSA-75, OVC-cOSA-31, and D17 cells, which were stimulated or not with 5 ng/mL TGFβ in the presence (+ Doxo) or absence (−Doxo) of Doxorubicin. Readings were first blank-corrected to fluorescence values obtained for the media only control. Bars depict the average ± SEM from two independent experiments plated in duplicate. Asterisks depict statistical differences as determined by a one-way ANOVA (Kruskal-Wallis test) with Dunn-Sidak correction to identify significant differences relative to control within each, the –Doxo and + Doxo treatment groups; * indicates p

    Journal: BMC Veterinary Research

    Article Title: An evaluation of TAZ and YAP crosstalk with TGFβ signalling in canine osteosarcoma suggests involvement of hippo signalling in disease progression

    doi: 10.1186/s12917-018-1651-5

    Figure Lengend Snippet: Effect of TAZ or YAP knockdown plus and minus TGFβ on cell viability of canine OSA cell lines. Graphs depict the fluorescence readings at 530/590 nm when TAZ ( a ) or YAP ( b ) was depleted in OVC-cOSA-75, OVC-cOSA-31, and D17 cells, which were stimulated or not with 5 ng/mL TGFβ in the presence (+ Doxo) or absence (−Doxo) of Doxorubicin. Readings were first blank-corrected to fluorescence values obtained for the media only control. Bars depict the average ± SEM from two independent experiments plated in duplicate. Asterisks depict statistical differences as determined by a one-way ANOVA (Kruskal-Wallis test) with Dunn-Sidak correction to identify significant differences relative to control within each, the –Doxo and + Doxo treatment groups; * indicates p

    Article Snippet: Antibodies and TGFβ Antibodies used in this study included: YAP (#14074), β actin (#4967S), pSmad2 (#3108), and Smad2 (#5339) or Smad2/3 (#5678) for pSmad2 normalization (all from Cell Signaling Technology, New England Biolabs, Ltd., Whitby, ON, Canada).

    Techniques: Fluorescence

    Effect of TAZ or YAP knockdown plus and minus TGFβ on cell proliferation of canine OSA cell lines. Representative immunofluorescence images of the mitotic marker phosphor-histone H3 (pHH3) taken at 20X objective lens for primary tumour-derived cell lines ( a ), and metastatic cell lines ( b ). Graphs depict the relative percentage of pHH3 positivity cells, as determined by dividing the number of pHH3 cells by the number of cells in the field of view. Five images were taken per experimental group and averaged. Error bars depict average ± SEM from three independent experiments. Scale bar = 100 μm. Asterisks depict statistical differences as determined by a two-way ANOVA with post-hoc Tukey-Kramer, * indicates p

    Journal: BMC Veterinary Research

    Article Title: An evaluation of TAZ and YAP crosstalk with TGFβ signalling in canine osteosarcoma suggests involvement of hippo signalling in disease progression

    doi: 10.1186/s12917-018-1651-5

    Figure Lengend Snippet: Effect of TAZ or YAP knockdown plus and minus TGFβ on cell proliferation of canine OSA cell lines. Representative immunofluorescence images of the mitotic marker phosphor-histone H3 (pHH3) taken at 20X objective lens for primary tumour-derived cell lines ( a ), and metastatic cell lines ( b ). Graphs depict the relative percentage of pHH3 positivity cells, as determined by dividing the number of pHH3 cells by the number of cells in the field of view. Five images were taken per experimental group and averaged. Error bars depict average ± SEM from three independent experiments. Scale bar = 100 μm. Asterisks depict statistical differences as determined by a two-way ANOVA with post-hoc Tukey-Kramer, * indicates p

    Article Snippet: Antibodies and TGFβ Antibodies used in this study included: YAP (#14074), β actin (#4967S), pSmad2 (#3108), and Smad2 (#5339) or Smad2/3 (#5678) for pSmad2 normalization (all from Cell Signaling Technology, New England Biolabs, Ltd., Whitby, ON, Canada).

    Techniques: Immunofluorescence, Marker, Derivative Assay

    Dose of SM16 to prevent increased, but not basal, transforming growth factor (Tgf)-β signaling in retinal vessels. Bar plot of phosphorylated (P)-Smad2/total Smad2 ratio in retinal microvessels in control rats (C), rats injected i.p. with human recombinant active TGF-β 1 (60 μg; R D Systems, Minneapolis, MN) and euthanized 1 hour later (C+TGFb); and rats treated for 5 days with SM16 at 6 mg/kg per day and then injected with TGF-β 1 (SM16+TGFb). Blots were probed for P-Smad2, stripped, and reprobed for total Smad2 for calculation of the ratio in each individual sample. Data are expressed as means ± SD. n = 2 (SM16+TGFb); n = 3 (C); n = 5 (C+TGFb). ∗ P

    Journal: The American Journal of Pathology

    Article Title: The Increased Transforming Growth Factor-β Signaling Induced by Diabetes Protects Retinal Vessels

    doi: 10.1016/j.ajpath.2016.11.007

    Figure Lengend Snippet: Dose of SM16 to prevent increased, but not basal, transforming growth factor (Tgf)-β signaling in retinal vessels. Bar plot of phosphorylated (P)-Smad2/total Smad2 ratio in retinal microvessels in control rats (C), rats injected i.p. with human recombinant active TGF-β 1 (60 μg; R D Systems, Minneapolis, MN) and euthanized 1 hour later (C+TGFb); and rats treated for 5 days with SM16 at 6 mg/kg per day and then injected with TGF-β 1 (SM16+TGFb). Blots were probed for P-Smad2, stripped, and reprobed for total Smad2 for calculation of the ratio in each individual sample. Data are expressed as means ± SD. n = 2 (SM16+TGFb); n = 3 (C); n = 5 (C+TGFb). ∗ P

    Article Snippet: To measure Tgf-β signaling, membranes were probed with antibodies to phosphorylated-Smad2 (catalog number 3101, 1:1000; Cell Signaling, Danvers, MA) and, after stripping, antibodies to total Smad2 (catalog number 07-408, 1:1000; Millipore, Billerica, MA).

    Techniques: Injection, Recombinant

    QSG inhibited activation of TGF-β1/Smad3 through reducing the recruitment of M1 macrophages in myocardial tissue. (A) IF staining showed that splenectomy reduced the recruitment of M1 macrophages in myocardial tissue; QSG treatment decreased the recruitment of M1 macrophages. (B) Masson and Sirius Red staining showed that QSG inhibited cardiac fibrosis. (C) Western blot showed that QSG down-regulated the expressions of AT1, MCP-1 and CCR2 in myocardial tissue compared with that in the model group. (D) Western blot showed that QSG decreased the expressions of TGF-β1, Smad3, collagen III and MMP2 in cardiac tissue. All data were presented as means ± SD from independent experiments performed in triplicate. # P

    Journal: Frontiers in Pharmacology

    Article Title: Qishen Granule Improved Cardiac Remodeling via Balancing M1 and M2 Macrophages

    doi: 10.3389/fphar.2019.01399

    Figure Lengend Snippet: QSG inhibited activation of TGF-β1/Smad3 through reducing the recruitment of M1 macrophages in myocardial tissue. (A) IF staining showed that splenectomy reduced the recruitment of M1 macrophages in myocardial tissue; QSG treatment decreased the recruitment of M1 macrophages. (B) Masson and Sirius Red staining showed that QSG inhibited cardiac fibrosis. (C) Western blot showed that QSG down-regulated the expressions of AT1, MCP-1 and CCR2 in myocardial tissue compared with that in the model group. (D) Western blot showed that QSG decreased the expressions of TGF-β1, Smad3, collagen III and MMP2 in cardiac tissue. All data were presented as means ± SD from independent experiments performed in triplicate. # P

    Article Snippet: After electrophoresis at 100 V for 1–1.5 h, proteins were transferred to PVDF membranes at 300 mA for 1–1.5 h. Afterwards, the membrane blocked with 5% skim milk for 1–1.5 h at room temperature, incubated on a shaker, and washed with TBS-T. Western blot analysis was conducted using anti-AT1 (ab18801; Abcam, United States), anti-MCP-1 (ab25124, Abcam, United States), anti-CCR2 (PAI-27409), anti-TGF-β1 (3711s, Cell Signaling Technology, Germany), anti-Smad3 (ab28379, Abcam, United States), anti-MMP2 (ab86607, Abcam, United States), anti-Col III (ab7778, Abcam, United States), anti-VEGF (ab10972, Abcam, United States), anti-CD31 (ab24590, Abcam, United States) and anti-GAPDH (ab8245, Abcam, United States) at 4°C overnight.

    Techniques: Activation Assay, Staining, Western Blot

    β -catenin/Foxo diverts TGF- β signaling from a profibrotic to an anti-inflammatory pathway. Diagram depicting the mechanism by which TGF- β signaling can be diverted from profibrotic to anti-inflammatory by shifting β -catenin from TCF to Foxo binding (symbols and arrows in red).

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Redirecting TGF-β Signaling through the β-Catenin/Foxo Complex Prevents Kidney Fibrosis

    doi: 10.1681/ASN.2016121362

    Figure Lengend Snippet: β -catenin/Foxo diverts TGF- β signaling from a profibrotic to an anti-inflammatory pathway. Diagram depicting the mechanism by which TGF- β signaling can be diverted from profibrotic to anti-inflammatory by shifting β -catenin from TCF to Foxo binding (symbols and arrows in red).

    Article Snippet: Immunoblot analysis was performed with anti– β -catenin (1:1000; BD Biosciences), anti-Foxo1 (1:1000), anti-TCF (1:1000), or anti–TGF- β (1:100; Cell Signaling).

    Techniques: Binding Assay

    Shift of β -catenin from TCF to Foxo inhibits inflammatory and increases anti-inflammatory cytokine production via TGF- β iTregs in UUO mice. (A) Three days after UUO operation, splenocytes were stimulated with phorbol 12-myristate 13-acetate (5 ng/ml) and ionomycin (0.5 μ g/ml) in the absence of Golgi stop for 4 hours, and culture supernatants were analyzed for the production of cytokines (IL-6, TNF- α , IFN- γ , IL-17A, and IL-10) using cytometric bead assay ( n =6 per group per cytokine; one of two experiments is shown). (B) Serum was analyzed for the production of cytokines (IL-6, TNF- α , IFN- γ , IL-17A, and IL-10) using cytometric bead assay ( n =6 per group per cytokine; one of two experiments is shown). Statistical significance was determined by one-way ANOVA followed by Tukey post hoc test. All data are expressed as the means±SEM. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Redirecting TGF-β Signaling through the β-Catenin/Foxo Complex Prevents Kidney Fibrosis

    doi: 10.1681/ASN.2016121362

    Figure Lengend Snippet: Shift of β -catenin from TCF to Foxo inhibits inflammatory and increases anti-inflammatory cytokine production via TGF- β iTregs in UUO mice. (A) Three days after UUO operation, splenocytes were stimulated with phorbol 12-myristate 13-acetate (5 ng/ml) and ionomycin (0.5 μ g/ml) in the absence of Golgi stop for 4 hours, and culture supernatants were analyzed for the production of cytokines (IL-6, TNF- α , IFN- γ , IL-17A, and IL-10) using cytometric bead assay ( n =6 per group per cytokine; one of two experiments is shown). (B) Serum was analyzed for the production of cytokines (IL-6, TNF- α , IFN- γ , IL-17A, and IL-10) using cytometric bead assay ( n =6 per group per cytokine; one of two experiments is shown). Statistical significance was determined by one-way ANOVA followed by Tukey post hoc test. All data are expressed as the means±SEM. * P

    Article Snippet: Immunoblot analysis was performed with anti– β -catenin (1:1000; BD Biosciences), anti-Foxo1 (1:1000), anti-TCF (1:1000), or anti–TGF- β (1:100; Cell Signaling).

    Techniques: Mouse Assay

    Shift of β -catenin from TCF to Foxo increases TGF- β iTregs. (A) TGF- β 1 expression in the obstructed kidney of UUO mice on days 1, 3, 5, 7, and 14. Representative immunoblots of total kidney protein lysates showing increased expression of TGF- β 1 protein. The bar graph shows TGF- β 1 band density normalized to β -actin. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc test ( n =6 per group). * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Redirecting TGF-β Signaling through the β-Catenin/Foxo Complex Prevents Kidney Fibrosis

    doi: 10.1681/ASN.2016121362

    Figure Lengend Snippet: Shift of β -catenin from TCF to Foxo increases TGF- β iTregs. (A) TGF- β 1 expression in the obstructed kidney of UUO mice on days 1, 3, 5, 7, and 14. Representative immunoblots of total kidney protein lysates showing increased expression of TGF- β 1 protein. The bar graph shows TGF- β 1 band density normalized to β -actin. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc test ( n =6 per group). * P

    Article Snippet: Immunoblot analysis was performed with anti– β -catenin (1:1000; BD Biosciences), anti-Foxo1 (1:1000), anti-TCF (1:1000), or anti–TGF- β (1:100; Cell Signaling).

    Techniques: Expressing, Mouse Assay, Western Blot

    β -catenin controls Foxp3 expression by binding to Foxo1 in TGF- β iTregs. (A) Flow cytometry analysis of Foxp3 in rhTGF- β 1 iTregs arising from CD4 + CD25 − T cells. (B) Representative histogram analyses of Foxp3 in rhTGF- β 1 iTregs arising from EL4 cells transfected with constructs of β -catenin or F-Trcp-Ecad ( β -catenin degradation chimera). (C) Representative histogram analyses of Foxp3 in rhTGF- β 1 iTregs arising from EL4 cells treated with LiCl or ICG-001. MFI, mean fluorescence intensity. (D) Lysates from nuclear extracts of EL4 cells immunoprecipitated with anti-IgG, anti-Foxo1, or anti-TCF antibodies and analyzed by Western blot using an anti– β -catenin antibody. IP, immunoprecipitation; WB, Western blot. (E) ChIP results for Foxo1 association with Foxp3 regulatory elements in rhTGF- β 1–induced EL4 cells after treatment with plasmids of β -catenin or F-TrCP-Ecad transfection or with ICG-001. Quantitative ChIP data are presented. Data were normalized against input DNA and are presented as fold change. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Redirecting TGF-β Signaling through the β-Catenin/Foxo Complex Prevents Kidney Fibrosis

    doi: 10.1681/ASN.2016121362

    Figure Lengend Snippet: β -catenin controls Foxp3 expression by binding to Foxo1 in TGF- β iTregs. (A) Flow cytometry analysis of Foxp3 in rhTGF- β 1 iTregs arising from CD4 + CD25 − T cells. (B) Representative histogram analyses of Foxp3 in rhTGF- β 1 iTregs arising from EL4 cells transfected with constructs of β -catenin or F-Trcp-Ecad ( β -catenin degradation chimera). (C) Representative histogram analyses of Foxp3 in rhTGF- β 1 iTregs arising from EL4 cells treated with LiCl or ICG-001. MFI, mean fluorescence intensity. (D) Lysates from nuclear extracts of EL4 cells immunoprecipitated with anti-IgG, anti-Foxo1, or anti-TCF antibodies and analyzed by Western blot using an anti– β -catenin antibody. IP, immunoprecipitation; WB, Western blot. (E) ChIP results for Foxo1 association with Foxp3 regulatory elements in rhTGF- β 1–induced EL4 cells after treatment with plasmids of β -catenin or F-TrCP-Ecad transfection or with ICG-001. Quantitative ChIP data are presented. Data were normalized against input DNA and are presented as fold change. * P

    Article Snippet: Immunoblot analysis was performed with anti– β -catenin (1:1000; BD Biosciences), anti-Foxo1 (1:1000), anti-TCF (1:1000), or anti–TGF- β (1:100; Cell Signaling).

    Techniques: Expressing, Binding Assay, Flow Cytometry, Cytometry, Transfection, Construct, Fluorescence, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation

    miR-584-5p promoted cellular senescence and negatively regulated TGFβ signaling pathway via downregulation of WWP1. a . miR-584-5p up-regulation or WWP1-shRNA promoted cellular senescence by detecting SA-β-gal activity. The rescue experiments for miR-584-5p overexpression were performed by ectopic expression of WWP1 without its 3’-UTR in MGC803 cells. b . Similar rescue experiments for miR-584-5p silencing was performed by down-regulation of WWP1 in SGC7901 cells. c . The expression levels of senescence-related proteins were evaluated by Western blot. d . Expression of p53 signifcantly increased by miR-584-5p was ameliorated by the p53 inhibitor pifthrin-α. e . Treatment of pifthrin-α signifcantly receded miR-584-5p induced apoptosis of the MGC803 cells which was measured in a flow cytometer. f . Positive SA-β-Gal stained cells induced by miR-584-5p was confined by treatment of pifthrin-α. h . Levels of key proteins involved in TGFβ signaling by western blot. i . Levels of key proteins involved in TGFβ signaling in MGC803 cells treated with LY364947 were measured by western blot. j . The expression level of TGFβ was determined in MGC803 and SGC7901 by immunofluorescence assays. Scale bar is 25 μm. * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Overexpression of miR-584-5p inhibits proliferation and induces apoptosis by targeting WW domain-containing E3 ubiquitin protein ligase 1 in gastric cancer

    doi: 10.1186/s13046-017-0532-2

    Figure Lengend Snippet: miR-584-5p promoted cellular senescence and negatively regulated TGFβ signaling pathway via downregulation of WWP1. a . miR-584-5p up-regulation or WWP1-shRNA promoted cellular senescence by detecting SA-β-gal activity. The rescue experiments for miR-584-5p overexpression were performed by ectopic expression of WWP1 without its 3’-UTR in MGC803 cells. b . Similar rescue experiments for miR-584-5p silencing was performed by down-regulation of WWP1 in SGC7901 cells. c . The expression levels of senescence-related proteins were evaluated by Western blot. d . Expression of p53 signifcantly increased by miR-584-5p was ameliorated by the p53 inhibitor pifthrin-α. e . Treatment of pifthrin-α signifcantly receded miR-584-5p induced apoptosis of the MGC803 cells which was measured in a flow cytometer. f . Positive SA-β-Gal stained cells induced by miR-584-5p was confined by treatment of pifthrin-α. h . Levels of key proteins involved in TGFβ signaling by western blot. i . Levels of key proteins involved in TGFβ signaling in MGC803 cells treated with LY364947 were measured by western blot. j . The expression level of TGFβ was determined in MGC803 and SGC7901 by immunofluorescence assays. Scale bar is 25 μm. * p

    Article Snippet: The cells were rinsed then with PBS three times and permeabilized with 0.2% Triton X-100 for 10 min. To block non-specific binding, the cells were incubated with PBS containing 1% BSA for 30 min and then incubated with the primary antibody TGFβ (Cell Signaling Technology, 1:1,000) at 4 °C.

    Techniques: shRNA, Activity Assay, Over Expression, Expressing, Western Blot, Flow Cytometry, Cytometry, Staining, Immunofluorescence

    Expression of the TGFβ type I receptor (TGFβ-RI) and type II receptor (TGFβ-RII) mRNA and proteins in parental IEC-6 and IEC-Cdx2L1 cells treated with 4 mM IPTG for 12 and 16 days. A: representative PCR-amplified products displayed in agarose gels for TGFβ-RI (177 bp) and TGFβ-RII (503 bp). Total cellular RNA was harvested at various times after IPTG treatment and the mRNA levels for TGFβ-RI and TGFβ-RII were determined by semiquantitative RT-PCR analysis. B: representative auto radiograms of Western blots from cells described in A. Whole cell lysates were harvested, applied to each lane (50 μg) equally, and subjected to electrophoresis on 10% acrylamide gel. Levels of TGFβ-RI (∼52 kDa) and TGFβ-RII (∼75 kDa) were identified by probing nitrocellulose with the specific antibodies. After the blot was stripped, actin (∼42 kDa) immunoblotting was performed as an internal control for equal loading. C: quantitative analysis of Western blots by densitometry from cells described in B. a, TGFβ-RI; b, TGFβ-RII. Values are means ± SE of data from 3 separate experiments; relative levels of TGFβ-RI and TGFβ-RII were corrected for loading as measured by densitometry of actin. *P

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Differentiated intestinal epithelial cells express high levels of TGF-? receptors and exhibit increased sensitivity to growth inhibition

    doi:

    Figure Lengend Snippet: Expression of the TGFβ type I receptor (TGFβ-RI) and type II receptor (TGFβ-RII) mRNA and proteins in parental IEC-6 and IEC-Cdx2L1 cells treated with 4 mM IPTG for 12 and 16 days. A: representative PCR-amplified products displayed in agarose gels for TGFβ-RI (177 bp) and TGFβ-RII (503 bp). Total cellular RNA was harvested at various times after IPTG treatment and the mRNA levels for TGFβ-RI and TGFβ-RII were determined by semiquantitative RT-PCR analysis. B: representative auto radiograms of Western blots from cells described in A. Whole cell lysates were harvested, applied to each lane (50 μg) equally, and subjected to electrophoresis on 10% acrylamide gel. Levels of TGFβ-RI (∼52 kDa) and TGFβ-RII (∼75 kDa) were identified by probing nitrocellulose with the specific antibodies. After the blot was stripped, actin (∼42 kDa) immunoblotting was performed as an internal control for equal loading. C: quantitative analysis of Western blots by densitometry from cells described in B. a, TGFβ-RI; b, TGFβ-RII. Values are means ± SE of data from 3 separate experiments; relative levels of TGFβ-RI and TGFβ-RII were corrected for loading as measured by densitometry of actin. *P

    Article Snippet: Anti-TGFβ-RI and RII antibodies were from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Electrophoresis, Acrylamide Gel Assay

    Relative levels of TGFβ-RI and TGFβ-RII mRNAs in control differentiated cells and cells treated with either α-difluoromethylornithine (DFMO) alone or DFMO plus spermidine (SPD). Relative levels of TGFβ-RI (A) and RII (B) mRNAs (arbitrary units). Differentiated cells were grown in DMEM containing 5% dFBS in the presence or absence of DFMO (5 mM) or DFMO + SPD (5 μM) for 4 and 6 days. The relative levels of TGFβ-RI and TGFβ-RII mRNAs were determined by semi-quantitative RT-PCR analysis. Data was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [optical density (OD) of the TGFβ-RI or RII mRNA/OD of the GAPDH] and expressed as means ± SE of data from 3 separate experiments. *P

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Differentiated intestinal epithelial cells express high levels of TGF-? receptors and exhibit increased sensitivity to growth inhibition

    doi:

    Figure Lengend Snippet: Relative levels of TGFβ-RI and TGFβ-RII mRNAs in control differentiated cells and cells treated with either α-difluoromethylornithine (DFMO) alone or DFMO plus spermidine (SPD). Relative levels of TGFβ-RI (A) and RII (B) mRNAs (arbitrary units). Differentiated cells were grown in DMEM containing 5% dFBS in the presence or absence of DFMO (5 mM) or DFMO + SPD (5 μM) for 4 and 6 days. The relative levels of TGFβ-RI and TGFβ-RII mRNAs were determined by semi-quantitative RT-PCR analysis. Data was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [optical density (OD) of the TGFβ-RI or RII mRNA/OD of the GAPDH] and expressed as means ± SE of data from 3 separate experiments. *P

    Article Snippet: Anti-TGFβ-RI and RII antibodies were from Cell Signaling Technology (Danvers, MA).

    Techniques: Quantitative RT-PCR

    Effect of short-term treatment with IPTG on TGFβ-RI and TGFβ-RII expression in stable IEC-Cdx2L1 cells. A: Representative PCR-amplified products of TGFβ-RI and TGFβ-RII. After cells were cultured in growth DMEM medium for 2 days, IPTG was added to the medium at a final concentration of 4 mM. Total cellular RNA was isolated 24 and 48 h after addition of IPTG and mRNA levels for TGFβ-RI and TGFβ-RII were measured. B: Quantitative analysis of data described in A. Values are means ± SE of data from 3 separate experiments.

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Differentiated intestinal epithelial cells express high levels of TGF-? receptors and exhibit increased sensitivity to growth inhibition

    doi:

    Figure Lengend Snippet: Effect of short-term treatment with IPTG on TGFβ-RI and TGFβ-RII expression in stable IEC-Cdx2L1 cells. A: Representative PCR-amplified products of TGFβ-RI and TGFβ-RII. After cells were cultured in growth DMEM medium for 2 days, IPTG was added to the medium at a final concentration of 4 mM. Total cellular RNA was isolated 24 and 48 h after addition of IPTG and mRNA levels for TGFβ-RI and TGFβ-RII were measured. B: Quantitative analysis of data described in A. Values are means ± SE of data from 3 separate experiments.

    Article Snippet: Anti-TGFβ-RI and RII antibodies were from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, Cell Culture, Concentration Assay, Isolation

    CAFs activated TGF- β /Smad signalling of breast cancer cells through secreted TGF- β 1. ( A ) TGF- β 1, TGF- β 2 and TGF- β 3 secreted by CAFs in CAF-CM were detected by cytokine antibody array. Cancer-associated fibroblasts secreted significantly more TGF- β 1 than TGF- β 2 and TGF- β 3. ( B ) TGFB1 expression in CAFs and breast cancer cell lines was detected by reverse transcription (RT)-quantitative PCR (QPCR). Cancer-associated fibroblasts expressed significantly higher TGFB1 expression levels than breast cancer cell lines MCF-7, T47D or MDA-MB-231. ( C ) The expression of TGF- β RII, total Smad2 and phosphorylated Smad2 in breast cancer cell lines cultured with CAF-CM was detected by immunoblotting. Phosphorylated Smad2 expression levels in MCF-7, T47D and MDA-MB-231 cells cultured with CAF-CM were significantly higher than their control cells, whereas total Smad2 and TGF- β RII were not significantly changed. ( D ) TGFB1 expression in breast cancer cells cultured with CAF-CM was detected by RT-QPCR. TGFB1 expression levels in MCF-7, T47D and MDA-MB-231 cell lines were not significantly affected by culturing in CAF-CM.

    Journal: British Journal of Cancer

    Article Title: Cancer-associated fibroblasts induce epithelial–mesenchymal transition of breast cancer cells through paracrine TGF-β signalling

    doi: 10.1038/bjc.2013.768

    Figure Lengend Snippet: CAFs activated TGF- β /Smad signalling of breast cancer cells through secreted TGF- β 1. ( A ) TGF- β 1, TGF- β 2 and TGF- β 3 secreted by CAFs in CAF-CM were detected by cytokine antibody array. Cancer-associated fibroblasts secreted significantly more TGF- β 1 than TGF- β 2 and TGF- β 3. ( B ) TGFB1 expression in CAFs and breast cancer cell lines was detected by reverse transcription (RT)-quantitative PCR (QPCR). Cancer-associated fibroblasts expressed significantly higher TGFB1 expression levels than breast cancer cell lines MCF-7, T47D or MDA-MB-231. ( C ) The expression of TGF- β RII, total Smad2 and phosphorylated Smad2 in breast cancer cell lines cultured with CAF-CM was detected by immunoblotting. Phosphorylated Smad2 expression levels in MCF-7, T47D and MDA-MB-231 cells cultured with CAF-CM were significantly higher than their control cells, whereas total Smad2 and TGF- β RII were not significantly changed. ( D ) TGFB1 expression in breast cancer cells cultured with CAF-CM was detected by RT-QPCR. TGFB1 expression levels in MCF-7, T47D and MDA-MB-231 cell lines were not significantly affected by culturing in CAF-CM.

    Article Snippet: The primary antibodies used were anti-Vimentin, anti-E-cadherin, anti-TGF-β RII (K105; Cell Signaling Technology, Danvers, Massachusetts, USA), anti-Smad2 (86F7; Cell Signaling Technology) and anti-pSmad2 (Phospho-Ser245/250/255; Cell Signaling Technology) at a dilution of 1 : 1000, β -actin (Sigma-Aldrich, St Louis, MO, USA) at a dilution of 1 : 3000, and the secondary antibody (GE Healthcare, Piscataway, NJ, USA) was used at a dilution of 1 : 2500.

    Techniques: Ab Array, Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Cell Culture, Quantitative RT-PCR

    TGF-β signaling pathway was down-regulated after treating with SB431542. a Collagen of ovaries slices was stained by Sirius red (Left is Vehicle group, and right is SB431542 treated group). By immunohistochemical analysis, downregulation of TGF-β ( b ) and p-Smad3 ( c ) was observed in SB431542 group compared to vehicle-control rats. d Vehicle exposed (the top) and SB431542 treated (the bottom). PCOS-like rat ovary tissue sections were IHC stained for a-SMA. (×100). e The expression of vehicle treated and SB431542 treated rat ovarian TGF-β (left) and Smad3 (middle) and p-Smad3 (right) protein were measured relative to GAPDH levels. f The expression of vehicle treated and SB431542 treated rat ovarian Smad2 (left) and p-Smad2 (right) protein were measured relative to GAPDH levels. g a-SMA and MMP2 protein expression in vehicle and SB431542 treated rat ovaries was tested by Western blot. (n = 6 in each group. The results are presented as mean ± SD of three independent experiments. *p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.002, “→” is directed in the direction of fibrosis. “▲” is directed in the direction of a-SMA; A, albuginea)

    Journal: Journal of Ovarian Research

    Article Title: DHEA-induced ovarian hyperfibrosis is mediated by TGF-β signaling pathway

    doi: 10.1186/s13048-017-0375-7

    Figure Lengend Snippet: TGF-β signaling pathway was down-regulated after treating with SB431542. a Collagen of ovaries slices was stained by Sirius red (Left is Vehicle group, and right is SB431542 treated group). By immunohistochemical analysis, downregulation of TGF-β ( b ) and p-Smad3 ( c ) was observed in SB431542 group compared to vehicle-control rats. d Vehicle exposed (the top) and SB431542 treated (the bottom). PCOS-like rat ovary tissue sections were IHC stained for a-SMA. (×100). e The expression of vehicle treated and SB431542 treated rat ovarian TGF-β (left) and Smad3 (middle) and p-Smad3 (right) protein were measured relative to GAPDH levels. f The expression of vehicle treated and SB431542 treated rat ovarian Smad2 (left) and p-Smad2 (right) protein were measured relative to GAPDH levels. g a-SMA and MMP2 protein expression in vehicle and SB431542 treated rat ovaries was tested by Western blot. (n = 6 in each group. The results are presented as mean ± SD of three independent experiments. *p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.002, “→” is directed in the direction of fibrosis. “▲” is directed in the direction of a-SMA; A, albuginea)

    Article Snippet: Immunohistochemistry (IHC) Four μm tissue sections (ovaries), were first incubated with specific antibodies against TGF-β (3711S, Cell Signaling Technology, CST), P-Smad3 (#9520S, Cell Signaling Technology, CST), a-SMA (ab32575, Abcam), and Collagen IV (Abcam), overnight at 4 °C.

    Techniques: Staining, Immunohistochemistry, Expressing, Western Blot

    TGF-β-induced fibrosis signaling pathway

    Journal: Journal of Ovarian Research

    Article Title: DHEA-induced ovarian hyperfibrosis is mediated by TGF-β signaling pathway

    doi: 10.1186/s13048-017-0375-7

    Figure Lengend Snippet: TGF-β-induced fibrosis signaling pathway

    Article Snippet: Immunohistochemistry (IHC) Four μm tissue sections (ovaries), were first incubated with specific antibodies against TGF-β (3711S, Cell Signaling Technology, CST), P-Smad3 (#9520S, Cell Signaling Technology, CST), a-SMA (ab32575, Abcam), and Collagen IV (Abcam), overnight at 4 °C.

    Techniques:

    Immunohistochemical analysis of TGF-β and Smads/p-Smads in Oil + DHEA-induced rats ovarian, and the detection of the expression of TGF-β and p-Smad3. a Immunohistochemical analysis of TGF-β and the density mean; b p-Smad3 and the density mean. c Percentage of TGF-β protein in rat ovaries (left) were determined by western blot and quantified by RT-PCR (middle) at least three times. d p-Smad3 and Smad3 protein levels. e Smad2/p-Smad2 protein levels. n = 6 in each group. Mean ± S.D.; * p ≤ 0.05

    Journal: Journal of Ovarian Research

    Article Title: DHEA-induced ovarian hyperfibrosis is mediated by TGF-β signaling pathway

    doi: 10.1186/s13048-017-0375-7

    Figure Lengend Snippet: Immunohistochemical analysis of TGF-β and Smads/p-Smads in Oil + DHEA-induced rats ovarian, and the detection of the expression of TGF-β and p-Smad3. a Immunohistochemical analysis of TGF-β and the density mean; b p-Smad3 and the density mean. c Percentage of TGF-β protein in rat ovaries (left) were determined by western blot and quantified by RT-PCR (middle) at least three times. d p-Smad3 and Smad3 protein levels. e Smad2/p-Smad2 protein levels. n = 6 in each group. Mean ± S.D.; * p ≤ 0.05

    Article Snippet: Immunohistochemistry (IHC) Four μm tissue sections (ovaries), were first incubated with specific antibodies against TGF-β (3711S, Cell Signaling Technology, CST), P-Smad3 (#9520S, Cell Signaling Technology, CST), a-SMA (ab32575, Abcam), and Collagen IV (Abcam), overnight at 4 °C.

    Techniques: Immunohistochemistry, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Expression of TGF-β downstream signaling molecules. a-b Levels of ovarian CTGF and fibronectin proteins (left) and mRNA (middle), detected by western blotting and quantitative-PCR assay. c mRNA levels of MMP2/MMP9 in Oil + DHEA-induced rats compared with Oil-treated rats. a-SMA is highly expressed in DHEA-induced rats. Oil + DHEA-induced rat ovaries ( e ) show a signally proliferation of a-SMA compared with Oil-treated rat ovaries ( d ). ×100. Ovarian protein was harvested to measure a-SMA expression ( f ) . n = 6 in each group. Data are mean ± S.D.; * p ≤ 0.05

    Journal: Journal of Ovarian Research

    Article Title: DHEA-induced ovarian hyperfibrosis is mediated by TGF-β signaling pathway

    doi: 10.1186/s13048-017-0375-7

    Figure Lengend Snippet: Expression of TGF-β downstream signaling molecules. a-b Levels of ovarian CTGF and fibronectin proteins (left) and mRNA (middle), detected by western blotting and quantitative-PCR assay. c mRNA levels of MMP2/MMP9 in Oil + DHEA-induced rats compared with Oil-treated rats. a-SMA is highly expressed in DHEA-induced rats. Oil + DHEA-induced rat ovaries ( e ) show a signally proliferation of a-SMA compared with Oil-treated rat ovaries ( d ). ×100. Ovarian protein was harvested to measure a-SMA expression ( f ) . n = 6 in each group. Data are mean ± S.D.; * p ≤ 0.05

    Article Snippet: Immunohistochemistry (IHC) Four μm tissue sections (ovaries), were first incubated with specific antibodies against TGF-β (3711S, Cell Signaling Technology, CST), P-Smad3 (#9520S, Cell Signaling Technology, CST), a-SMA (ab32575, Abcam), and Collagen IV (Abcam), overnight at 4 °C.

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

    Andro attenuated HSC activation through inhibition of the TGF- β 1/Smad2 signaling pathway. (a) Representative immunofluorescent staining of α -SMA. (b) The mRNA levels of α -SMA were measured by q-PCR. (c) The protein expression of α -SMA, TGF- β 1, p-Smad2, p-Smad3, and Smad2/3 was examined by Western blot. n = 3. ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Andrographolide Ameliorates Liver Fibrosis in Mice: Involvement of TLR4/NF-κB and TGF-β1/Smad2 Signaling Pathways

    doi: 10.1155/2018/7808656

    Figure Lengend Snippet: Andro attenuated HSC activation through inhibition of the TGF- β 1/Smad2 signaling pathway. (a) Representative immunofluorescent staining of α -SMA. (b) The mRNA levels of α -SMA were measured by q-PCR. (c) The protein expression of α -SMA, TGF- β 1, p-Smad2, p-Smad3, and Smad2/3 was examined by Western blot. n = 3. ∗∗ p

    Article Snippet: Anti-TGF- β 1, anti-p-Smad2, anti-pSmad3, and anti-Smad2/3 antibodies were from Cell Signaling Technology Inc. (Beverly, MA, USA).

    Techniques: Activation Assay, Inhibition, Staining, Polymerase Chain Reaction, Expressing, Western Blot

    Andrographolide (Andro) improved CCl 4 -induced liver fibrosis in mice. (a) Representative histology of Sirius Red and immunohistochemical staining of α -SMA and TGF- β 1. (b–d) Quantification of positive staining areas was measured by ImageJ software. (e) Hepatic hydroxyproline content. (f) The protein expression of α -SMA and TGF- β 1 was examined by Western blot. (g, h) Serum levels of ALT and AST. n = 6. ### p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Andrographolide Ameliorates Liver Fibrosis in Mice: Involvement of TLR4/NF-κB and TGF-β1/Smad2 Signaling Pathways

    doi: 10.1155/2018/7808656

    Figure Lengend Snippet: Andrographolide (Andro) improved CCl 4 -induced liver fibrosis in mice. (a) Representative histology of Sirius Red and immunohistochemical staining of α -SMA and TGF- β 1. (b–d) Quantification of positive staining areas was measured by ImageJ software. (e) Hepatic hydroxyproline content. (f) The protein expression of α -SMA and TGF- β 1 was examined by Western blot. (g, h) Serum levels of ALT and AST. n = 6. ### p

    Article Snippet: Anti-TGF- β 1, anti-p-Smad2, anti-pSmad3, and anti-Smad2/3 antibodies were from Cell Signaling Technology Inc. (Beverly, MA, USA).

    Techniques: Mouse Assay, Immunohistochemistry, Staining, Software, Expressing, Western Blot, AST Assay

    Cytokine concentrations in one-way MLR supernatants according to flow cytometric bead arrays ((a) IFN- γ ; (b) TNF- α ; (c) IL-10; (d) IL-4) and ELISA ((e) TGF- β ) presented as scatter plots with mean bar. “∗” P

    Journal: Clinical and Developmental Immunology

    Article Title: Vascularized Composite Allograft Rejection Is Delayed by Intrajejunal Treatment with Donor Splenocytes without Concomitant Immunosuppressants

    doi: 10.1155/2012/704063

    Figure Lengend Snippet: Cytokine concentrations in one-way MLR supernatants according to flow cytometric bead arrays ((a) IFN- γ ; (b) TNF- α ; (c) IL-10; (d) IL-4) and ELISA ((e) TGF- β ) presented as scatter plots with mean bar. “∗” P

    Article Snippet: ELISA for Quantifying TGF-β Concentrations TGF-β concentration was quantified in 96 hr MLR supernatants and Day +7 sera by ELISA with specific antibody to TGF-β according to the manufacturer's protocol (Biosource International; Catalog no. KAC1688/KAC1689).

    Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay

    Cytokine concentrations according to flow-CBA ((a) IFN- γ ; (b) TNF- α ; (c) IL-10; (d) IL-4) and ELISA ((e) TGF- β ) in Day +7 sera from TREATED/VCA and SHAM/VCA Groups presented as scatter plots with mean bar. “∗” P

    Journal: Clinical and Developmental Immunology

    Article Title: Vascularized Composite Allograft Rejection Is Delayed by Intrajejunal Treatment with Donor Splenocytes without Concomitant Immunosuppressants

    doi: 10.1155/2012/704063

    Figure Lengend Snippet: Cytokine concentrations according to flow-CBA ((a) IFN- γ ; (b) TNF- α ; (c) IL-10; (d) IL-4) and ELISA ((e) TGF- β ) in Day +7 sera from TREATED/VCA and SHAM/VCA Groups presented as scatter plots with mean bar. “∗” P

    Article Snippet: ELISA for Quantifying TGF-β Concentrations TGF-β concentration was quantified in 96 hr MLR supernatants and Day +7 sera by ELISA with specific antibody to TGF-β according to the manufacturer's protocol (Biosource International; Catalog no. KAC1688/KAC1689).

    Techniques: Flow Cytometry, Crocin Bleaching Assay, Enzyme-linked Immunosorbent Assay

    Coculture of CD4 + CD25 + and CD4 + CD25 − T cells results in high level IL-10 production. CD4 + CD25 + and CD4 + CD25 − T cells were MACS ® sorted from PBMCs of healthy individuals. These cells were either cultured alone or at a 1:1 ratio and activated with platebound anti-CD3 and soluble anti-CD28 (10 μg/ml, respectively). (A) After various time points supernatants were analyzed for cytokine production by ELISA. IL-10 production peaked 48 h after onset of culture and was markedly higher in the coculture of CD4 + CD25 + and CD4 + CD25 − T cells than in the cultures of each of the cell types alone. A representative out of five independent standardized experiments is shown. No elevated levels of INF-α or TGF-β could be measured (data not shown). (B) The different T cell populations were also activated with mature allogeneic DCs (DC/T cell ratio 1:20) compared with anti-CD3 and anti-CD28 (10 μg/ml, respectively). Cytokines were measured 48 h after onset of culture. Results were similar in five independent experiments. (C) For the last 6 h of activation with anti-CD3 and anti-CD28 2 μM monensin was added to the cultures. Staining of CD4 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for intracellular IL-10 using PE-conjugated specific Abs. One of five independent experiments is shown.

    Journal: The Journal of Experimental Medicine

    Article Title: Human CD4+CD25+ Regulatory, Contact-dependent T Cells Induce Interleukin 10-producing, Contact-independent Type 1-like Regulatory T Cells

    doi: 10.1084/jem.20020642

    Figure Lengend Snippet: Coculture of CD4 + CD25 + and CD4 + CD25 − T cells results in high level IL-10 production. CD4 + CD25 + and CD4 + CD25 − T cells were MACS ® sorted from PBMCs of healthy individuals. These cells were either cultured alone or at a 1:1 ratio and activated with platebound anti-CD3 and soluble anti-CD28 (10 μg/ml, respectively). (A) After various time points supernatants were analyzed for cytokine production by ELISA. IL-10 production peaked 48 h after onset of culture and was markedly higher in the coculture of CD4 + CD25 + and CD4 + CD25 − T cells than in the cultures of each of the cell types alone. A representative out of five independent standardized experiments is shown. No elevated levels of INF-α or TGF-β could be measured (data not shown). (B) The different T cell populations were also activated with mature allogeneic DCs (DC/T cell ratio 1:20) compared with anti-CD3 and anti-CD28 (10 μg/ml, respectively). Cytokines were measured 48 h after onset of culture. Results were similar in five independent experiments. (C) For the last 6 h of activation with anti-CD3 and anti-CD28 2 μM monensin was added to the cultures. Staining of CD4 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for intracellular IL-10 using PE-conjugated specific Abs. One of five independent experiments is shown.

    Article Snippet: Cytokine analysis was performed at different time points by analysis of supernatants with commercially available ELISA kits for human IL-10, IFN-α (Biosource International), and TGF-β (BD PharMingen).

    Techniques: Magnetic Cell Separation, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Staining, Expressing

    Th17 cells expressing RANKL promote osteoclast maturation A. Naïve CD4+ T cells were differentiated into Th17 cells for 5 days and the surface expression of RANKL and CD4 and the intracellular expression of IL-17 were detected by flow cytometry. B. The osteoclast precursor cell line RAW 264.7 was cultured with or without RANKL (50 ng/ml) and SDF-1a (20ng/ml) for 3 days. Naïve CD4+ T cells were added to the cultures with or without Th17-skewing cytokines (IL-6, IL-23, and TGF-β). After 3 days, RAW 264.7 cells were fixed and stained for TRAP activity. Total TRAP-positive cells in the absence of RANKL and SDF-1α C . TRAP-positive multinucleated cells in the presence of RANKL and SDF-1α were counted. D . Data are presented as means ± SEM (* P

    Journal: Oncotarget

    Article Title: Mesenchymal stem cells inhibit RANK-RANKL interactions between osteoclasts and Th17 cells via osteoprotegerin activity

    doi: 10.18632/oncotarget.21379

    Figure Lengend Snippet: Th17 cells expressing RANKL promote osteoclast maturation A. Naïve CD4+ T cells were differentiated into Th17 cells for 5 days and the surface expression of RANKL and CD4 and the intracellular expression of IL-17 were detected by flow cytometry. B. The osteoclast precursor cell line RAW 264.7 was cultured with or without RANKL (50 ng/ml) and SDF-1a (20ng/ml) for 3 days. Naïve CD4+ T cells were added to the cultures with or without Th17-skewing cytokines (IL-6, IL-23, and TGF-β). After 3 days, RAW 264.7 cells were fixed and stained for TRAP activity. Total TRAP-positive cells in the absence of RANKL and SDF-1α C . TRAP-positive multinucleated cells in the presence of RANKL and SDF-1α were counted. D . Data are presented as means ± SEM (* P

    Article Snippet: To induce differentiation into Th17 cells, cells were stimulated with anti-CD3 antibody (2.5 g/ml, Biolegend, San Diego, CA, USA), anti-CD28 antibody (5 μg/ml, Biolegend), IL-6 (50 ng/ml, Biolegend), IL-23 (20 ng/ml, Biolegend), TGF-β (5 ng/ml, Biolegend), and anti-IFN-γ blocking antibody (100 ng/ml, Biolegend) for 6 days.

    Techniques: Expressing, Flow Cytometry, Cytometry, Cell Culture, Staining, Activity Assay

    Th17 cells induce osteoclastogenesis in the absence of RANKL stimulation A. RAW 264.7 cells were cultured on calcium phosphate-coated plates. Naïve CD4+ T cells were added to the RAW 264.7 cell cultures with anti-CD3 Ab, anti-CD28 Ab, IL-6, IL-23, and TGF-β. To test the effect of T-MSC-derived OPG, RAW 264.7 cell cultures were treated with naïve CD4+ T cells and T-CM, OPG-knockdown T-CM, control siRNA knockdown T-CM, or OPG-knockdown T-CM supplemented with rhOPG. After 6 days, resorption activity assays were performed. B. Intact pit areas were assessed using ImageJ software. Data are presented as means ± SEM (* P

    Journal: Oncotarget

    Article Title: Mesenchymal stem cells inhibit RANK-RANKL interactions between osteoclasts and Th17 cells via osteoprotegerin activity

    doi: 10.18632/oncotarget.21379

    Figure Lengend Snippet: Th17 cells induce osteoclastogenesis in the absence of RANKL stimulation A. RAW 264.7 cells were cultured on calcium phosphate-coated plates. Naïve CD4+ T cells were added to the RAW 264.7 cell cultures with anti-CD3 Ab, anti-CD28 Ab, IL-6, IL-23, and TGF-β. To test the effect of T-MSC-derived OPG, RAW 264.7 cell cultures were treated with naïve CD4+ T cells and T-CM, OPG-knockdown T-CM, control siRNA knockdown T-CM, or OPG-knockdown T-CM supplemented with rhOPG. After 6 days, resorption activity assays were performed. B. Intact pit areas were assessed using ImageJ software. Data are presented as means ± SEM (* P

    Article Snippet: To induce differentiation into Th17 cells, cells were stimulated with anti-CD3 antibody (2.5 g/ml, Biolegend, San Diego, CA, USA), anti-CD28 antibody (5 μg/ml, Biolegend), IL-6 (50 ng/ml, Biolegend), IL-23 (20 ng/ml, Biolegend), TGF-β (5 ng/ml, Biolegend), and anti-IFN-γ blocking antibody (100 ng/ml, Biolegend) for 6 days.

    Techniques: Cell Culture, Derivative Assay, Activity Assay, Software

    Th17 cells enhance osteoclast activity in the presence of RANKL stimulation A. RAW 264.7 cells were cultured on calcium phosphate-coated plates and stimulated with RANKL (50 ng/ml) and SDF-1 (20 ng/ml). Recombinant rhOPG (100 ng/ml), naïve CD4+ T cells (with anti-CD3 and anti-CD28 Abs), or Th17-skewing cytokines alone (IL-6, IL-23, and TGF-β) were added. B. RAW 264.7 cells were cultured with Th17 cells in the presence of RANKL (50 ng/ml) and SDF-1 (20 ng/ml). OPG-knockdown T-CM or control siRNA-treated T-CM were added to RAW 264.7 cell cultures to observe the effect of T-MSC-derived OPG. Exogenous rhOPG was used as a positive control and was also added to OPG-knockdown T-CM. C. - D. After 6 days, resorption activity assays were performed and the intact pit areas were assessed using ImageJ software. Data are presented as means ± SEM (* P

    Journal: Oncotarget

    Article Title: Mesenchymal stem cells inhibit RANK-RANKL interactions between osteoclasts and Th17 cells via osteoprotegerin activity

    doi: 10.18632/oncotarget.21379

    Figure Lengend Snippet: Th17 cells enhance osteoclast activity in the presence of RANKL stimulation A. RAW 264.7 cells were cultured on calcium phosphate-coated plates and stimulated with RANKL (50 ng/ml) and SDF-1 (20 ng/ml). Recombinant rhOPG (100 ng/ml), naïve CD4+ T cells (with anti-CD3 and anti-CD28 Abs), or Th17-skewing cytokines alone (IL-6, IL-23, and TGF-β) were added. B. RAW 264.7 cells were cultured with Th17 cells in the presence of RANKL (50 ng/ml) and SDF-1 (20 ng/ml). OPG-knockdown T-CM or control siRNA-treated T-CM were added to RAW 264.7 cell cultures to observe the effect of T-MSC-derived OPG. Exogenous rhOPG was used as a positive control and was also added to OPG-knockdown T-CM. C. - D. After 6 days, resorption activity assays were performed and the intact pit areas were assessed using ImageJ software. Data are presented as means ± SEM (* P

    Article Snippet: To induce differentiation into Th17 cells, cells were stimulated with anti-CD3 antibody (2.5 g/ml, Biolegend, San Diego, CA, USA), anti-CD28 antibody (5 μg/ml, Biolegend), IL-6 (50 ng/ml, Biolegend), IL-23 (20 ng/ml, Biolegend), TGF-β (5 ng/ml, Biolegend), and anti-IFN-γ blocking antibody (100 ng/ml, Biolegend) for 6 days.

    Techniques: Activity Assay, Cell Culture, Recombinant, Derivative Assay, Positive Control, Software

    miRNA-30e regulates Snai1/TGF-β/Nox4 protein expression. (A) Snai1, (B) TGF-β, (C) Smad2 and (D) Nox4 protein expression by statistical analysis, and (E) western blotting analysis. ## P

    Journal: International Journal of Molecular Medicine

    Article Title: MicroRNA-30e regulates TGF-β-mediated NADPH oxidase 4-dependent oxidative stress by Snai1 in atherosclerosis

    doi: 10.3892/ijmm.2019.4102

    Figure Lengend Snippet: miRNA-30e regulates Snai1/TGF-β/Nox4 protein expression. (A) Snai1, (B) TGF-β, (C) Smad2 and (D) Nox4 protein expression by statistical analysis, and (E) western blotting analysis. ## P

    Article Snippet: The membrane was blocked with 5% skim milk powder at room temperature for 2 h and incubated with Snai1 (cat. no. sc-271977; 1:1,000), TGF-β (cat. no. sc-52892; 1:1,000; both Santa Cruz Biotechnology, Inc.), Samd2 (cat. no. ab122028; 1:1,000), Nox4 (cat. no. ab133303; 1:1,000; both Abcam, Cambridge, USA) and GAPDH (cat. no. sc-293335; 1:5,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

    Techniques: Expressing, Western Blot

    miRNA-30e regulates TGF-β-mediated NADPH oxidase 4-dependent oxidative stress by Snai1. (A) Heat map for signaling pathway, (B) Snai1 was a putative target of miRNA-30e and (C) luciferase reporter activity, (D) Snai1 protein expression. (E) Snai1, (F) TGF-β, (G) Smad2 and (H) Nox4 protein expression by statistical analysis, and (I) western blotting analysis. ## P

    Journal: International Journal of Molecular Medicine

    Article Title: MicroRNA-30e regulates TGF-β-mediated NADPH oxidase 4-dependent oxidative stress by Snai1 in atherosclerosis

    doi: 10.3892/ijmm.2019.4102

    Figure Lengend Snippet: miRNA-30e regulates TGF-β-mediated NADPH oxidase 4-dependent oxidative stress by Snai1. (A) Heat map for signaling pathway, (B) Snai1 was a putative target of miRNA-30e and (C) luciferase reporter activity, (D) Snai1 protein expression. (E) Snai1, (F) TGF-β, (G) Smad2 and (H) Nox4 protein expression by statistical analysis, and (I) western blotting analysis. ## P

    Article Snippet: The membrane was blocked with 5% skim milk powder at room temperature for 2 h and incubated with Snai1 (cat. no. sc-271977; 1:1,000), TGF-β (cat. no. sc-52892; 1:1,000; both Santa Cruz Biotechnology, Inc.), Samd2 (cat. no. ab122028; 1:1,000), Nox4 (cat. no. ab133303; 1:1,000; both Abcam, Cambridge, USA) and GAPDH (cat. no. sc-293335; 1:5,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

    Techniques: Luciferase, Activity Assay, Expressing, Western Blot

    Activation of Snai1 reduces the effects of miRNA-30e on the oxidative stress in vitro model. (A) Snai1, (B) TGF-β, (C) Smad2 and (D) Nox4 protein expression by statistical analysis, and (E) western blotting analysis, (F) MDA, (G) SOD, (H) GSH and (I) GSH-PX, (J) ROS levels and (K) green fluorescent protein transfection. Scale bar, 100 µ m. ** P

    Journal: International Journal of Molecular Medicine

    Article Title: MicroRNA-30e regulates TGF-β-mediated NADPH oxidase 4-dependent oxidative stress by Snai1 in atherosclerosis

    doi: 10.3892/ijmm.2019.4102

    Figure Lengend Snippet: Activation of Snai1 reduces the effects of miRNA-30e on the oxidative stress in vitro model. (A) Snai1, (B) TGF-β, (C) Smad2 and (D) Nox4 protein expression by statistical analysis, and (E) western blotting analysis, (F) MDA, (G) SOD, (H) GSH and (I) GSH-PX, (J) ROS levels and (K) green fluorescent protein transfection. Scale bar, 100 µ m. ** P

    Article Snippet: The membrane was blocked with 5% skim milk powder at room temperature for 2 h and incubated with Snai1 (cat. no. sc-271977; 1:1,000), TGF-β (cat. no. sc-52892; 1:1,000; both Santa Cruz Biotechnology, Inc.), Samd2 (cat. no. ab122028; 1:1,000), Nox4 (cat. no. ab133303; 1:1,000; both Abcam, Cambridge, USA) and GAPDH (cat. no. sc-293335; 1:5,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

    Techniques: Activation Assay, In Vitro, Expressing, Western Blot, Multiple Displacement Amplification, Transfection

    Activation of TGF-β reduces the effects of miRNA-30e on the oxidative stress in vitro model. (A) TGF-β, (B) Smad2 and (C) Nox4 protein expression by statistical analysis and (D) western blotting analysis, (E) MDA, (F) SOD, (G) GSH and (H) GSH-PX (I) ROS levels and (J) green fluorescent protein. ** P

    Journal: International Journal of Molecular Medicine

    Article Title: MicroRNA-30e regulates TGF-β-mediated NADPH oxidase 4-dependent oxidative stress by Snai1 in atherosclerosis

    doi: 10.3892/ijmm.2019.4102

    Figure Lengend Snippet: Activation of TGF-β reduces the effects of miRNA-30e on the oxidative stress in vitro model. (A) TGF-β, (B) Smad2 and (C) Nox4 protein expression by statistical analysis and (D) western blotting analysis, (E) MDA, (F) SOD, (G) GSH and (H) GSH-PX (I) ROS levels and (J) green fluorescent protein. ** P

    Article Snippet: The membrane was blocked with 5% skim milk powder at room temperature for 2 h and incubated with Snai1 (cat. no. sc-271977; 1:1,000), TGF-β (cat. no. sc-52892; 1:1,000; both Santa Cruz Biotechnology, Inc.), Samd2 (cat. no. ab122028; 1:1,000), Nox4 (cat. no. ab133303; 1:1,000; both Abcam, Cambridge, USA) and GAPDH (cat. no. sc-293335; 1:5,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

    Techniques: Activation Assay, In Vitro, Expressing, Western Blot, Multiple Displacement Amplification

    Functional characterization of Treg after EP treatment. (A) CTLA-4 protein expression in CD25 + cells purified from the pool of lymphoid tissues, normalized to the expression of β-actin, along with the representative blot. (B) The proportion of TGF-β-expressing cells within Treg, along with representative dot plots. (C) IL-10 protein expression in CD25 + cells purified from a pool of lymphoid tissues, normalized to the expression of β-actin, along with the representative blot. (D) The proportion of IL-10-expressing Treg within pancreatic infiltrates, along with representative dot plots. (E) The level of inhibition of effector T cell (Teff–CD4 + CD25 − ) proliferation after co-culture with Treg (CD4 + CD25 + ) cells purified from a pool of lymphoid tissues of MLDS or MLDS+EP-treated mice. Proliferation was measured after 72 h of incubation by CFSE dilution (in Teff). (F) Proportion of divided Teff, or Teff cultured in the presence of Treg (1:1 ratio). Representative dot plots are given below. All measurements were performed on samples from 7 animals per group. * p

    Journal: Frontiers in Immunology

    Article Title: Ethyl Pyruvate Stimulates Regulatory T Cells and Ameliorates Type 1 Diabetes Development in Mice

    doi: 10.3389/fimmu.2018.03130

    Figure Lengend Snippet: Functional characterization of Treg after EP treatment. (A) CTLA-4 protein expression in CD25 + cells purified from the pool of lymphoid tissues, normalized to the expression of β-actin, along with the representative blot. (B) The proportion of TGF-β-expressing cells within Treg, along with representative dot plots. (C) IL-10 protein expression in CD25 + cells purified from a pool of lymphoid tissues, normalized to the expression of β-actin, along with the representative blot. (D) The proportion of IL-10-expressing Treg within pancreatic infiltrates, along with representative dot plots. (E) The level of inhibition of effector T cell (Teff–CD4 + CD25 − ) proliferation after co-culture with Treg (CD4 + CD25 + ) cells purified from a pool of lymphoid tissues of MLDS or MLDS+EP-treated mice. Proliferation was measured after 72 h of incubation by CFSE dilution (in Teff). (F) Proportion of divided Teff, or Teff cultured in the presence of Treg (1:1 ratio). Representative dot plots are given below. All measurements were performed on samples from 7 animals per group. * p

    Article Snippet: For Th1 differentiation the cells were additionally stimulated with IL-12 (20 ng/ml, R & D Systems, Minneapolis, MN, USA) and anti-IL-4 antibody (10 ng/ml, eBioscience), for Th17 differentiation the cells were stimulated with TGF-β (10 ng/ml, R & D Systems) and IL-6 (10 ng/ml, R & D Systems), while for Treg differentiation the cells were stimulated with either TGF-β (2 ng/ml) and IL-2 (10 ng/ml) (both from R & D Systems) (complete stimulation), or only with IL-2 (incomplete stimulation).

    Techniques: Functional Assay, Expressing, Purification, Inhibition, Co-Culture Assay, Mouse Assay, Incubation, Cell Culture

    In vitro effect of EP on Th cell differentiation. (A) The proportion of Th1 (CD4 + IFN-γ + ), Th17 (CD4 + IL-17 + ), and Treg (CD4 + CD25 high FoxP3 + ) cells after 96 h of incubation of CD4 + CD25 − cells with adequate stimulation (described in Material and methods) and in the presence of EP (125 μM), added 24 h after the start of cell cultivation. Representative dot plots are shown. Cells were first gated on live CD4 + , followed by the gate on IFN-γ + , IL-17 + or CD25 high FoxpP3 + . (B) The proportion of Treg that expressed GITR, CTLA-4, and PD-1, determined by flow cytometry. Representative dot plots show the first gate on live CTLA-4 + , GITR + or PD-1 + population, followed by the gate on CD4 + CD25 high . (C) The proportion of Treg after incomplete stimulation (anti-CD3+anti-CD28+IL-2) after 5 days of incubation of CD4 + cells in the presence or absence of EP. Representative dot plots show the first gate on live CD4 + cells, followed by the gate on CD25 high FoxpP3 + . (D) IL-10 concentration in supernatants of EP-treated CD4 + cells in the presence of complete Treg stimulation (anti-CD3/anti-CD28+IL-2+TGF-β), after 5 days of incubation, measured by ELISA. * p

    Journal: Frontiers in Immunology

    Article Title: Ethyl Pyruvate Stimulates Regulatory T Cells and Ameliorates Type 1 Diabetes Development in Mice

    doi: 10.3389/fimmu.2018.03130

    Figure Lengend Snippet: In vitro effect of EP on Th cell differentiation. (A) The proportion of Th1 (CD4 + IFN-γ + ), Th17 (CD4 + IL-17 + ), and Treg (CD4 + CD25 high FoxP3 + ) cells after 96 h of incubation of CD4 + CD25 − cells with adequate stimulation (described in Material and methods) and in the presence of EP (125 μM), added 24 h after the start of cell cultivation. Representative dot plots are shown. Cells were first gated on live CD4 + , followed by the gate on IFN-γ + , IL-17 + or CD25 high FoxpP3 + . (B) The proportion of Treg that expressed GITR, CTLA-4, and PD-1, determined by flow cytometry. Representative dot plots show the first gate on live CTLA-4 + , GITR + or PD-1 + population, followed by the gate on CD4 + CD25 high . (C) The proportion of Treg after incomplete stimulation (anti-CD3+anti-CD28+IL-2) after 5 days of incubation of CD4 + cells in the presence or absence of EP. Representative dot plots show the first gate on live CD4 + cells, followed by the gate on CD25 high FoxpP3 + . (D) IL-10 concentration in supernatants of EP-treated CD4 + cells in the presence of complete Treg stimulation (anti-CD3/anti-CD28+IL-2+TGF-β), after 5 days of incubation, measured by ELISA. * p

    Article Snippet: For Th1 differentiation the cells were additionally stimulated with IL-12 (20 ng/ml, R & D Systems, Minneapolis, MN, USA) and anti-IL-4 antibody (10 ng/ml, eBioscience), for Th17 differentiation the cells were stimulated with TGF-β (10 ng/ml, R & D Systems) and IL-6 (10 ng/ml, R & D Systems), while for Treg differentiation the cells were stimulated with either TGF-β (2 ng/ml) and IL-2 (10 ng/ml) (both from R & D Systems) (complete stimulation), or only with IL-2 (incomplete stimulation).

    Techniques: In Vitro, Cell Differentiation, Incubation, Flow Cytometry, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

    The effect of EP on Treg differentiation and proliferation in vivo . (A) IL-2 protein expression in PLN, along with the representative blot. (B) The proportion of non-Treg producers of TGF-β in the pancreatic infiltrates, along with representative dot plots (first gated on live non-CD25 high cells, followed by the gate on TGF-β + ). (C) The proportion of proliferating Treg (CD25 high Ki67 + ) in PLN and pancreatic infiltrates. Representative dot plots (first gated on live CD25 high cells, followed by the gate on Ki67 + ) for pancreatic infiltrates are shown. All measurements were performed on samples from 7 animals per group. * p

    Journal: Frontiers in Immunology

    Article Title: Ethyl Pyruvate Stimulates Regulatory T Cells and Ameliorates Type 1 Diabetes Development in Mice

    doi: 10.3389/fimmu.2018.03130

    Figure Lengend Snippet: The effect of EP on Treg differentiation and proliferation in vivo . (A) IL-2 protein expression in PLN, along with the representative blot. (B) The proportion of non-Treg producers of TGF-β in the pancreatic infiltrates, along with representative dot plots (first gated on live non-CD25 high cells, followed by the gate on TGF-β + ). (C) The proportion of proliferating Treg (CD25 high Ki67 + ) in PLN and pancreatic infiltrates. Representative dot plots (first gated on live CD25 high cells, followed by the gate on Ki67 + ) for pancreatic infiltrates are shown. All measurements were performed on samples from 7 animals per group. * p

    Article Snippet: For Th1 differentiation the cells were additionally stimulated with IL-12 (20 ng/ml, R & D Systems, Minneapolis, MN, USA) and anti-IL-4 antibody (10 ng/ml, eBioscience), for Th17 differentiation the cells were stimulated with TGF-β (10 ng/ml, R & D Systems) and IL-6 (10 ng/ml, R & D Systems), while for Treg differentiation the cells were stimulated with either TGF-β (2 ng/ml) and IL-2 (10 ng/ml) (both from R & D Systems) (complete stimulation), or only with IL-2 (incomplete stimulation).

    Techniques: In Vivo, Expressing

    Proposed mechanism of EP's beneficial effect in T1D. EP enhances the regulatory arm of the immune response during T1D pathogenesis. From pancreatic lymph nodes, tolDC (CD11c + CD11b − CD103 + ) migrate into the pancreatic islets and their proportion becomes significantly higher. Also, Treg (CD4 + CD25 high ) in the pancreatic lymph nodes increase their suppressive properties (CTLA-4, IL-10, and TGF-β) and migrate into the inflamed pancreatic islet (increased CD11a and CD62L expression and proportion of CXCR3 + ). In the islet, since Treg are already activated (CD44 + ), they proliferate (Ki67 + ), and are retained at the site (CD103 + ). Also, some of Treg express T-bet (increased proportion after EP treatment) and thereby inhibit T-bet + effector cells (CD25 med ) within the PLN and pancreatic infiltrates. Finally, EP may stimulate in vitro and in vivo differentiation of Treg through enhancement of TGF-β production. All these events result in the preservation of β-cell function (insulin production) and reduction of HMGB1 expression.

    Journal: Frontiers in Immunology

    Article Title: Ethyl Pyruvate Stimulates Regulatory T Cells and Ameliorates Type 1 Diabetes Development in Mice

    doi: 10.3389/fimmu.2018.03130

    Figure Lengend Snippet: Proposed mechanism of EP's beneficial effect in T1D. EP enhances the regulatory arm of the immune response during T1D pathogenesis. From pancreatic lymph nodes, tolDC (CD11c + CD11b − CD103 + ) migrate into the pancreatic islets and their proportion becomes significantly higher. Also, Treg (CD4 + CD25 high ) in the pancreatic lymph nodes increase their suppressive properties (CTLA-4, IL-10, and TGF-β) and migrate into the inflamed pancreatic islet (increased CD11a and CD62L expression and proportion of CXCR3 + ). In the islet, since Treg are already activated (CD44 + ), they proliferate (Ki67 + ), and are retained at the site (CD103 + ). Also, some of Treg express T-bet (increased proportion after EP treatment) and thereby inhibit T-bet + effector cells (CD25 med ) within the PLN and pancreatic infiltrates. Finally, EP may stimulate in vitro and in vivo differentiation of Treg through enhancement of TGF-β production. All these events result in the preservation of β-cell function (insulin production) and reduction of HMGB1 expression.

    Article Snippet: For Th1 differentiation the cells were additionally stimulated with IL-12 (20 ng/ml, R & D Systems, Minneapolis, MN, USA) and anti-IL-4 antibody (10 ng/ml, eBioscience), for Th17 differentiation the cells were stimulated with TGF-β (10 ng/ml, R & D Systems) and IL-6 (10 ng/ml, R & D Systems), while for Treg differentiation the cells were stimulated with either TGF-β (2 ng/ml) and IL-2 (10 ng/ml) (both from R & D Systems) (complete stimulation), or only with IL-2 (incomplete stimulation).

    Techniques: Expressing, In Vitro, In Vivo, Preserving, Cell Function Assay

    Expression of fibronectin in cells, tissues and tumours. ( a ) The morphology of 4T1 cells with and without TGF-β induction (15 ng ml −1 , 5 days). Images were taken by phase-contrast microscopy; all scale bars, 50 μm. ( b ) RT–PCR analysis demonstrates that treatment with TGF-β induces upregulation of fibronectin and downregulation of E-cadherin, which are characteristic features of EMT, data represent the mean±s.d., n =3. ( c ) Representative western blots showing fibronectin expression in normal tissues, and in primary and metastatic 4T1 breast tumours in Balb/c mice. ( d ) Densitometric analysis of the expression of fibronectin normalized to that of β-actin. Values represent mean±s.d., n =3.

    Journal: Nature Communications

    Article Title: MRI detection of breast cancer micrometastases with a fibronectin-targeting contrast agent

    doi: 10.1038/ncomms8984

    Figure Lengend Snippet: Expression of fibronectin in cells, tissues and tumours. ( a ) The morphology of 4T1 cells with and without TGF-β induction (15 ng ml −1 , 5 days). Images were taken by phase-contrast microscopy; all scale bars, 50 μm. ( b ) RT–PCR analysis demonstrates that treatment with TGF-β induces upregulation of fibronectin and downregulation of E-cadherin, which are characteristic features of EMT, data represent the mean±s.d., n =3. ( c ) Representative western blots showing fibronectin expression in normal tissues, and in primary and metastatic 4T1 breast tumours in Balb/c mice. ( d ) Densitometric analysis of the expression of fibronectin normalized to that of β-actin. Values represent mean±s.d., n =3.

    Article Snippet: D-Luciferin (Gold Biotechnology, St Louis, MO), TGF-β (Abcam, Cambridge, MA), RNeasy Plus Kit (Qiagen,Valencia, CA), High Capacity cDNA Transcription Kit (Applied Biosystems, Waltham, MA), Mastercycler ep realplex2 (VWR, Radnor, PA), homogenization buffer, protease inhibitor cocktail (Sigma-Aldrich; P8340), SDS–PAGE gels, polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), anti-fibronectin polyclonal antibody (Abcam; 1:4000), anti-β-actin antibody (Cell Signaling Technology, Danvers, MA; 1:1000), rhodamine-red-X-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:200) and other related reagents were purchased and used according to the manufacturers' instructions.

    Techniques: Expressing, Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mouse Assay

    Targeting fibrin–fibronectin complexes for molecular MRI of breast cancer micrometastases. ( a ) Cancer cells from the primary tumour invade into distant organs through epithelial-to-mesenchymal transition (EMT) and transmit signals to prepare ‘soil' of ‘pre-metastatic niche' for metastases. The expression of fibronectin and its associated complexes, such as the fibrin–fibronectin complex, is upregulated by TGF-β. ( b ) The abundant fibrin–fibronectin complexes in the tumour ECM allow the binding of enough CREKA-Tris(Gd-DOTA) 3 to the ECM marker so as to generate sufficient signal enhancement for effective molecular MRI of small high-risk breast cancer and micrometastases.

    Journal: Nature Communications

    Article Title: MRI detection of breast cancer micrometastases with a fibronectin-targeting contrast agent

    doi: 10.1038/ncomms8984

    Figure Lengend Snippet: Targeting fibrin–fibronectin complexes for molecular MRI of breast cancer micrometastases. ( a ) Cancer cells from the primary tumour invade into distant organs through epithelial-to-mesenchymal transition (EMT) and transmit signals to prepare ‘soil' of ‘pre-metastatic niche' for metastases. The expression of fibronectin and its associated complexes, such as the fibrin–fibronectin complex, is upregulated by TGF-β. ( b ) The abundant fibrin–fibronectin complexes in the tumour ECM allow the binding of enough CREKA-Tris(Gd-DOTA) 3 to the ECM marker so as to generate sufficient signal enhancement for effective molecular MRI of small high-risk breast cancer and micrometastases.

    Article Snippet: D-Luciferin (Gold Biotechnology, St Louis, MO), TGF-β (Abcam, Cambridge, MA), RNeasy Plus Kit (Qiagen,Valencia, CA), High Capacity cDNA Transcription Kit (Applied Biosystems, Waltham, MA), Mastercycler ep realplex2 (VWR, Radnor, PA), homogenization buffer, protease inhibitor cocktail (Sigma-Aldrich; P8340), SDS–PAGE gels, polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), anti-fibronectin polyclonal antibody (Abcam; 1:4000), anti-β-actin antibody (Cell Signaling Technology, Danvers, MA; 1:1000), rhodamine-red-X-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:200) and other related reagents were purchased and used according to the manufacturers' instructions.

    Techniques: Magnetic Resonance Imaging, Expressing, Binding Assay, Marker

    Representative image of morphological changes in HPMCs. (Upper left) HPMCs cultured in control medium. (Upper middle) HPMCs cultured in medium containing 100 μg/ml of PSK. (Upper right) HPMCs cultured in medium containing 500 μg/ml of PSK. (Lower left) HPMCs cultured in medium containing 10 ng/ml of TGF-β. (Lower middle) HPMCs cultured in 10 ng/ml of TGF-β and 100 μg/ml of PSK. (Lower right) HPMCs cultured in 10 ng/ml of TGF-β and 500 μg/ml of PSK. HPMCs cultured in each condition for 72 h were visualized by phase contrast microscopy at magnification, ×200. HPMCs, human peritoneal mesothelial cells; PSK, protein-bound polysaccharide K.

    Journal: Oncology Reports

    Article Title: Protein-bound polysaccharide K suppresses tumor fibrosis in gastric cancer by inhibiting the TGF-β signaling pathway

    doi: 10.3892/or.2014.3636

    Figure Lengend Snippet: Representative image of morphological changes in HPMCs. (Upper left) HPMCs cultured in control medium. (Upper middle) HPMCs cultured in medium containing 100 μg/ml of PSK. (Upper right) HPMCs cultured in medium containing 500 μg/ml of PSK. (Lower left) HPMCs cultured in medium containing 10 ng/ml of TGF-β. (Lower middle) HPMCs cultured in 10 ng/ml of TGF-β and 100 μg/ml of PSK. (Lower right) HPMCs cultured in 10 ng/ml of TGF-β and 500 μg/ml of PSK. HPMCs cultured in each condition for 72 h were visualized by phase contrast microscopy at magnification, ×200. HPMCs, human peritoneal mesothelial cells; PSK, protein-bound polysaccharide K.

    Article Snippet: Co. (Japan) and TGF-β was purchased from Sigma-Aldrich, Inc. (USA).

    Techniques: Cell Culture, Microscopy

    Representative photomicrographs of HPMCs, labeled with antibodies to E-cadherin (red) and α-SMA (green). TGF-β-treated HPMCs showed increased expression of α-SMA, whereas both TGF-β and PSK-treated HPMCs showed almost equal expression of α-SMA when compared with the control. HPMCs, human peritoneal mesothelial cells; PSK, protein-bound polysaccharide K.

    Journal: Oncology Reports

    Article Title: Protein-bound polysaccharide K suppresses tumor fibrosis in gastric cancer by inhibiting the TGF-β signaling pathway

    doi: 10.3892/or.2014.3636

    Figure Lengend Snippet: Representative photomicrographs of HPMCs, labeled with antibodies to E-cadherin (red) and α-SMA (green). TGF-β-treated HPMCs showed increased expression of α-SMA, whereas both TGF-β and PSK-treated HPMCs showed almost equal expression of α-SMA when compared with the control. HPMCs, human peritoneal mesothelial cells; PSK, protein-bound polysaccharide K.

    Article Snippet: Co. (Japan) and TGF-β was purchased from Sigma-Aldrich, Inc. (USA).

    Techniques: Labeling, Expressing

    Induction of TAMs and CAFs by IL-6 in human bladder cancer tissues and confirmation by in vitro experiments. (A) Representative expression status for IL-6 in human bladder cancer tissues. Images were captured at 200× magnification. (B) The interrelationship between the percentage of IL-6-positive cancer cells and the number of tumor-infiltrating TAMs was examined using Spearman’s correlation. Spearman r was found to be 0.68 (95% confidence interval, 058–0.76). (C) Comparison of the percentage of IL-6-positive cancer with the induction level of CAFs (CAF score). (D) Morphological changes of THP-1 cells by PMA and IL-4 treatment. THP-1 cells were treated with 200 nM PMA for 24 h to differentiate them into resting macrophages (middle), followed by treatment with IL-4 (20 ng/mL) for 48 h (right). (E) Western blot analysis for confirming the generation of TAMs and CAFs. THP-1 cells were treated with a combination of PMA and 20 ng/mL of CXCL1, IL-6, or TGF-β, separately. Upregulation of CD204 indicates differentiation into TAMs. NIH3T3 cells were treated with 20 ng/mL of CXCL1, IL-6, or TGF-β, separately. Upregulation of αSMA indicates the activation of fibroblasts, which are known to be CAFs.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: CXCL1-Mediated Interaction of Cancer Cells with Tumor-Associated Macrophages and Cancer-Associated Fibroblasts Promotes Tumor Progression in Human Bladder Cancer

    doi: 10.1016/j.neo.2016.08.002

    Figure Lengend Snippet: Induction of TAMs and CAFs by IL-6 in human bladder cancer tissues and confirmation by in vitro experiments. (A) Representative expression status for IL-6 in human bladder cancer tissues. Images were captured at 200× magnification. (B) The interrelationship between the percentage of IL-6-positive cancer cells and the number of tumor-infiltrating TAMs was examined using Spearman’s correlation. Spearman r was found to be 0.68 (95% confidence interval, 058–0.76). (C) Comparison of the percentage of IL-6-positive cancer with the induction level of CAFs (CAF score). (D) Morphological changes of THP-1 cells by PMA and IL-4 treatment. THP-1 cells were treated with 200 nM PMA for 24 h to differentiate them into resting macrophages (middle), followed by treatment with IL-4 (20 ng/mL) for 48 h (right). (E) Western blot analysis for confirming the generation of TAMs and CAFs. THP-1 cells were treated with a combination of PMA and 20 ng/mL of CXCL1, IL-6, or TGF-β, separately. Upregulation of CD204 indicates differentiation into TAMs. NIH3T3 cells were treated with 20 ng/mL of CXCL1, IL-6, or TGF-β, separately. Upregulation of αSMA indicates the activation of fibroblasts, which are known to be CAFs.

    Article Snippet: Recombinant proteins, CXCL1 (R & D Biosystems, Minneapolis, MN, USA), TGF-β (Wako Pure Chemical, Osaka, Japan), IL-4, and IL-6 (Prospec, East Brunswick, NJ, USA) were used in the described experiments.

    Techniques: In Vitro, Expressing, Western Blot, Activation Assay

    MG induces YAP co-transcriptional activity in breast cancer cells. ( A ) Western blot detection of YAP in MDA-MB-231 cells under the indication conditions. Immunoblot is representative of three independent experiments. ( B ) Western blot of Phospho-Smad2/3 and Smad2/3 in MDA-MB-231 treated with MG until confluence and then with TGF-β during 2 hr. β-actin is used for normalization. ( C and D ) YAP mRNA and protein level assessed by qRT-PCR and Western blot, respectively, in MDA-MB-231 cells silenced for YAP (siYAP#1 and #2) and treated in the same conditions as in Figure 5D . Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. ( E ) YAP (Cell Signaling, 4912) and TEAD1 IF co-localization in MDA-MB-231 cells cultured under low (Sparse) density used as positive control and in high-density cultured cells (Confluent) in presence of MG. Magnification 630x. Data are representative of two independent experiments. ( F ) Proliferation assay on GLO1 -depleted MDA-MB-231 (sh GLO1 #1 and #2) silenced or not for YAP (siYAP#1 and #2) at different time points. Data (72 hr) were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. All immunoblots are representative of three independent experiments. ***p

    Journal: eLife

    Article Title: Methylglyoxal, a glycolysis side-product, induces Hsp90 glycation and YAP-mediated tumor growth and metastasis

    doi: 10.7554/eLife.19375

    Figure Lengend Snippet: MG induces YAP co-transcriptional activity in breast cancer cells. ( A ) Western blot detection of YAP in MDA-MB-231 cells under the indication conditions. Immunoblot is representative of three independent experiments. ( B ) Western blot of Phospho-Smad2/3 and Smad2/3 in MDA-MB-231 treated with MG until confluence and then with TGF-β during 2 hr. β-actin is used for normalization. ( C and D ) YAP mRNA and protein level assessed by qRT-PCR and Western blot, respectively, in MDA-MB-231 cells silenced for YAP (siYAP#1 and #2) and treated in the same conditions as in Figure 5D . Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. ( E ) YAP (Cell Signaling, 4912) and TEAD1 IF co-localization in MDA-MB-231 cells cultured under low (Sparse) density used as positive control and in high-density cultured cells (Confluent) in presence of MG. Magnification 630x. Data are representative of two independent experiments. ( F ) Proliferation assay on GLO1 -depleted MDA-MB-231 (sh GLO1 #1 and #2) silenced or not for YAP (siYAP#1 and #2) at different time points. Data (72 hr) were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. All immunoblots are representative of three independent experiments. ***p

    Article Snippet: TGF-β was obtained from Roche (Mannheim, Germany.

    Techniques: Activity Assay, Western Blot, Multiple Displacement Amplification, Quantitative RT-PCR, Cell Culture, Positive Control, Proliferation Assay

    The serum cytokine levels of TGF-β, IL-6 and IL-23 was measured by ELISA in MS patients and healthy controls. Comparison of TGF-β (A), IL-6 (B), IL-23 (C) cytokines concentrations between HC, RRMS patients in relapse and remission phase, SPMS and PPMS patients. Pearson’s correlation and regression tests between TGF-β (D), IL-6 (E), IL-23 (F) and Tc17 cells in MS patients. One-way ANOVA was used to test for differences between the groups. Subsequent multiple comparison pairwise test was Bonferroni. * P-value

    Journal: PLoS ONE

    Article Title: Differential Frequency of CD8+ T Cell Subsets in Multiple Sclerosis Patients with Various Clinical Patterns

    doi: 10.1371/journal.pone.0159565

    Figure Lengend Snippet: The serum cytokine levels of TGF-β, IL-6 and IL-23 was measured by ELISA in MS patients and healthy controls. Comparison of TGF-β (A), IL-6 (B), IL-23 (C) cytokines concentrations between HC, RRMS patients in relapse and remission phase, SPMS and PPMS patients. Pearson’s correlation and regression tests between TGF-β (D), IL-6 (E), IL-23 (F) and Tc17 cells in MS patients. One-way ANOVA was used to test for differences between the groups. Subsequent multiple comparison pairwise test was Bonferroni. * P-value

    Article Snippet: The concentrations of IL-6 (BD Biosciences, US), IL-23 (e-Bioscience, US) and TGF-β (USCN Life Sciences Inc. HK) were assessed by ELISA following the manufacturer’s instructions.

    Techniques: Enzyme-linked Immunosorbent Assay, Mass Spectrometry

    qRT-PCR analysis of selected genes in DLE lesional and normal skin. a – k RNA expression of general macrophage markers (CD163 and CD68) ( a , b ), M1 macrophage-related markers (CD127, TNF-α, CXCL10, STAT1, and CCL5) ( c – g ), and M2 macrophage-related markers (TGF-β, CD206, CD209, and arginase-1) ( h – k ) was compared in DLE lesional (n = 17) and normal (n = 12) skin via qRT-PCR. Multiple general and M1 macrophage-related markers were upregulated in DLE lesional skin. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. CCL chemokine (C-C motif) ligand, CXCL chemokine (C-X-C motif) ligand, DLE discoid lupus erythematosus, qRT-PCR quantitative real-time polymerase chain reaction, STAT signal transducer and activator of transcription, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha

    Journal: Arthritis Research & Therapy

    Article Title: A subset of CD163+ macrophages displays mixed polarizations in discoid lupus skin

    doi: 10.1186/s13075-015-0839-3

    Figure Lengend Snippet: qRT-PCR analysis of selected genes in DLE lesional and normal skin. a – k RNA expression of general macrophage markers (CD163 and CD68) ( a , b ), M1 macrophage-related markers (CD127, TNF-α, CXCL10, STAT1, and CCL5) ( c – g ), and M2 macrophage-related markers (TGF-β, CD206, CD209, and arginase-1) ( h – k ) was compared in DLE lesional (n = 17) and normal (n = 12) skin via qRT-PCR. Multiple general and M1 macrophage-related markers were upregulated in DLE lesional skin. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. CCL chemokine (C-C motif) ligand, CXCL chemokine (C-X-C motif) ligand, DLE discoid lupus erythematosus, qRT-PCR quantitative real-time polymerase chain reaction, STAT signal transducer and activator of transcription, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha

    Article Snippet: Slides were then incubated with rabbit anti-human primary antibodies—CD127 (Abcam, Cambridge, MA, USA), CD209 (Abcam), CXCL10 (PeproTech, Rocky Hill, NJ, USA), tumor necrosis factor-α (TNF-α) (Novus, Littleton, CO, USA), and TGF-β (Novus)—overnight followed by PBS washes.

    Techniques: Quantitative RT-PCR, RNA Expression, Real-time Polymerase Chain Reaction

    Double immunofluorescence staining for CD163 + macrophages and CD3 + T cells and selected macrophage markers in DLE lesional skin (n = 6–8). a – e DLE lesional skin was stained with both antibodies to CD163 ( red ) and selected macrophage markers ( green ), including CXCL10 ( a ), CD127 ( b ), TNF-α ( c ), TGF-β ( d ), and CD209 ( e ). Accompanying control slides are present on the upper right. Yellow arrowheads indicate example areas of overlap. A minority of CD163 + macrophages co-stained with these proteins, and the most overlap was seen with CD209. ( f ) DLE lesional skin was also stained with antibodies to CD3 ( red ) and CD127 ( green ). A subset of CD3 + T cells showed expression of CD127. Dotted line denotes epidermal-dermal junction. Total magnification: ×200. DLE discoid lupus erythematosus, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha

    Journal: Arthritis Research & Therapy

    Article Title: A subset of CD163+ macrophages displays mixed polarizations in discoid lupus skin

    doi: 10.1186/s13075-015-0839-3

    Figure Lengend Snippet: Double immunofluorescence staining for CD163 + macrophages and CD3 + T cells and selected macrophage markers in DLE lesional skin (n = 6–8). a – e DLE lesional skin was stained with both antibodies to CD163 ( red ) and selected macrophage markers ( green ), including CXCL10 ( a ), CD127 ( b ), TNF-α ( c ), TGF-β ( d ), and CD209 ( e ). Accompanying control slides are present on the upper right. Yellow arrowheads indicate example areas of overlap. A minority of CD163 + macrophages co-stained with these proteins, and the most overlap was seen with CD209. ( f ) DLE lesional skin was also stained with antibodies to CD3 ( red ) and CD127 ( green ). A subset of CD3 + T cells showed expression of CD127. Dotted line denotes epidermal-dermal junction. Total magnification: ×200. DLE discoid lupus erythematosus, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha

    Article Snippet: Slides were then incubated with rabbit anti-human primary antibodies—CD127 (Abcam, Cambridge, MA, USA), CD209 (Abcam), CXCL10 (PeproTech, Rocky Hill, NJ, USA), tumor necrosis factor-α (TNF-α) (Novus, Littleton, CO, USA), and TGF-β (Novus)—overnight followed by PBS washes.

    Techniques: Double Immunofluorescence Staining, Staining, Expressing

    Mast cell expression of IL-6 in parenchyma in patients with CF (A), and mast cell expression of TGF-β in parenchyma in IPF patients (B) . Overall significance for total mast cell expression in different parenchyma is shown in each picture. Significance to healthy controls is shown as: p

    Journal: Respiratory Research

    Article Title: Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis

    doi: 10.1186/1465-9921-12-139

    Figure Lengend Snippet: Mast cell expression of IL-6 in parenchyma in patients with CF (A), and mast cell expression of TGF-β in parenchyma in IPF patients (B) . Overall significance for total mast cell expression in different parenchyma is shown in each picture. Significance to healthy controls is shown as: p

    Article Snippet: After antigen retrieval and a blocking step with 5% horse serum, the IL-6 (mouse anti-IL-6, 1:50, Novocastra) or TGF-β (mouse anti-TGF-β, 1:40, Novocastra) immunoreactivity was visualised with a horse anti-mouse biotinylated secondary antibody and Alexa-Flour 555-conjugated streptavidin (Molecular Probes, Oregon, USA).

    Techniques: Expressing

    Expression patterns of interleukin (IL)-6 (A-C), and TGF-β (D-F) in small airways, pulmonary vessels, and alveolar parenchyma . In each graph, results are shown as the percentage of total mast cells, and of MC TC and MC T subtypes that are positive for the respective mediator. Data are expressed as medians with interquartile ranges. Statistical differences to never-smoking controls were analysed using Mann-Whitney and asterisks show significant difference where * denotes p

    Journal: Respiratory Research

    Article Title: Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis

    doi: 10.1186/1465-9921-12-139

    Figure Lengend Snippet: Expression patterns of interleukin (IL)-6 (A-C), and TGF-β (D-F) in small airways, pulmonary vessels, and alveolar parenchyma . In each graph, results are shown as the percentage of total mast cells, and of MC TC and MC T subtypes that are positive for the respective mediator. Data are expressed as medians with interquartile ranges. Statistical differences to never-smoking controls were analysed using Mann-Whitney and asterisks show significant difference where * denotes p

    Article Snippet: After antigen retrieval and a blocking step with 5% horse serum, the IL-6 (mouse anti-IL-6, 1:50, Novocastra) or TGF-β (mouse anti-TGF-β, 1:40, Novocastra) immunoreactivity was visualised with a horse anti-mouse biotinylated secondary antibody and Alexa-Flour 555-conjugated streptavidin (Molecular Probes, Oregon, USA).

    Techniques: Expressing, MANN-WHITNEY

    Identification of endoglin as a direct target of miR‐342‐5p. A, The 3′ UTR of human and mouse endoglin mRNA were aligned with Hsa‐miR‐342‐5p and Mmu‐miR‐342‐5p. Complementary sequences were marked in red. B and C, HUVEC s were transfected with miR‐342‐5p, miR‐342‐5p ASO , or control. The level of miR‐342‐5p was determined with qRT ‐ PCR (B). Endoglin expression was determined by using Western blot (C). Bands were quantitatively compared between groups. D, Total RNA was prepared from HUVEC s transfected with miR‐342‐5p or retina tissues derived from pups at postnatal day 7 that had accepted intravitreal injection of miR‐342‐5p on postnatal day 3. Endoglin mRNA level was determined by using qRT ‐ PCR . E, Reporter assay. HeLa cells were cotransfected with pGL 3‐endoglin wild type or pGL 3‐endoglin mutant, together with increasing amount of miR‐342‐5p. Cells were harvested 24 hours after the transfection, and luciferase activity was analyzed. F, Liver sinusoid endothelial cells were isolated from endothelial‐specific Notch‐activating mice and control mice, and expression of endoglin was determined by using qRT ‐ PCR . G, HUVEC s transfected with miR‐342‐5p or control were cultured in the presence of TGF ‐β for 30 minutes. The phosphorylation of Smad1/5 and Smad2/3 was determined by using Western blot and quantitatively compared. H, HUVEC s were transfected with control, miR‐342‐5p or miR‐342‐5p plus endoglin and stimulated with vascular endothelial growth factor. The total and phosphorylated Akt were determined using Western blot. Bars indicate mean± SD (n=5), * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: miR‐342‐5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor β Signaling

    doi: 10.1161/JAHA.115.003042

    Figure Lengend Snippet: Identification of endoglin as a direct target of miR‐342‐5p. A, The 3′ UTR of human and mouse endoglin mRNA were aligned with Hsa‐miR‐342‐5p and Mmu‐miR‐342‐5p. Complementary sequences were marked in red. B and C, HUVEC s were transfected with miR‐342‐5p, miR‐342‐5p ASO , or control. The level of miR‐342‐5p was determined with qRT ‐ PCR (B). Endoglin expression was determined by using Western blot (C). Bands were quantitatively compared between groups. D, Total RNA was prepared from HUVEC s transfected with miR‐342‐5p or retina tissues derived from pups at postnatal day 7 that had accepted intravitreal injection of miR‐342‐5p on postnatal day 3. Endoglin mRNA level was determined by using qRT ‐ PCR . E, Reporter assay. HeLa cells were cotransfected with pGL 3‐endoglin wild type or pGL 3‐endoglin mutant, together with increasing amount of miR‐342‐5p. Cells were harvested 24 hours after the transfection, and luciferase activity was analyzed. F, Liver sinusoid endothelial cells were isolated from endothelial‐specific Notch‐activating mice and control mice, and expression of endoglin was determined by using qRT ‐ PCR . G, HUVEC s transfected with miR‐342‐5p or control were cultured in the presence of TGF ‐β for 30 minutes. The phosphorylation of Smad1/5 and Smad2/3 was determined by using Western blot and quantitatively compared. H, HUVEC s were transfected with control, miR‐342‐5p or miR‐342‐5p plus endoglin and stimulated with vascular endothelial growth factor. The total and phosphorylated Akt were determined using Western blot. Bars indicate mean± SD (n=5), * P

    Article Snippet: The γ‐secretase inhibitor (Alexis Biochemicals), VEGF, and TGF‐β (Promega) were used at concentrations of 25, 30, and 2.5 ng/mL, respectively.

    Techniques: Transfection, Allele-specific Oligonucleotide, Quantitative RT-PCR, Expressing, Western Blot, Derivative Assay, Injection, Reporter Assay, Mutagenesis, Luciferase, Activity Assay, Isolation, Mouse Assay, Cell Culture

    miR‐342‐5p antagonized TGF ‐β in endothelial cells. A, HUVEC s were cultured in the presence of TGF ‐β or PBS , and the expression of miR‐342‐5p and EVL mRNA was determined using quantitative reverse transcription polymerase chain reaction. B, HUVEC s were transfected with miR‐342‐5p ASO or control and cultured in the presence of TGF ‐β or PBS . Cells were trypsinized 72 hours after transfection and tested for lumen formation, and the number of branches and the length (×10 3 μm) of cell cords of the enclosed lumens were measured. C, Cells in (B) were tested for sprouting by using the fibrin gel beads assay. Images were captured under a microscope and sprouting was quantitatively analyzed by measuring the number and length (×10 2 μm) of sprouts. D, Cells in (B) were assayed for the protein levels of CD 31, α‐ SMA , β‐catenin, and vimentin 72 hours after the transfection by using Western blot. Bars indicate mean± SD (n=3), * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: miR‐342‐5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor β Signaling

    doi: 10.1161/JAHA.115.003042

    Figure Lengend Snippet: miR‐342‐5p antagonized TGF ‐β in endothelial cells. A, HUVEC s were cultured in the presence of TGF ‐β or PBS , and the expression of miR‐342‐5p and EVL mRNA was determined using quantitative reverse transcription polymerase chain reaction. B, HUVEC s were transfected with miR‐342‐5p ASO or control and cultured in the presence of TGF ‐β or PBS . Cells were trypsinized 72 hours after transfection and tested for lumen formation, and the number of branches and the length (×10 3 μm) of cell cords of the enclosed lumens were measured. C, Cells in (B) were tested for sprouting by using the fibrin gel beads assay. Images were captured under a microscope and sprouting was quantitatively analyzed by measuring the number and length (×10 2 μm) of sprouts. D, Cells in (B) were assayed for the protein levels of CD 31, α‐ SMA , β‐catenin, and vimentin 72 hours after the transfection by using Western blot. Bars indicate mean± SD (n=3), * P

    Article Snippet: The γ‐secretase inhibitor (Alexis Biochemicals), VEGF, and TGF‐β (Promega) were used at concentrations of 25, 30, and 2.5 ng/mL, respectively.

    Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Allele-specific Oligonucleotide, Microscopy, Western Blot

    miR‐342‐5p inhibited vascularization in CNV . A, Healthy adult mice underwent laser‐induced CNV . The choroid membranes of the mice were collected on days 3 and 7 after laser injury, and the expression of miR‐342‐5p and EVL mRNA was compared with the control group. B, Mice underwent laser‐induced CNV and were injected intravitreally with miR‐342‐5p agomir. The level of miR‐342‐5p was determined with quantitative reverse transcription polymerase chain reaction. C, Healthy adult mice underwent laser‐induced CNV . miR‐342‐5p agomir or control was injected intravitreally 24 hours after laser burns. Choroid membranes were collected on day 7 and stained with isolectin B4. The relative CNV areas were compared between the 2 groups. D, A model showing that miR‐342‐5p functions as a multieffect repressor of angiogenesis in EC s. The expression of miR‐342‐5p is downregulated by VEGFR 2 signaling through Akt and upregulated by Notch signaling and TGF ‐β treatment. This mi RNA represses angiogenesis by suppressing VEGFR and endoglin‐mediated TGF ‐β receptor signaling, upregulating Dll4, and downregulating jagged 1 in Notch signaling, leading to decreased EC proliferation, migration, and probably increased endothelial–mesenchymal transition. Bars indicate mean± SD (n=5), * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: miR‐342‐5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor β Signaling

    doi: 10.1161/JAHA.115.003042

    Figure Lengend Snippet: miR‐342‐5p inhibited vascularization in CNV . A, Healthy adult mice underwent laser‐induced CNV . The choroid membranes of the mice were collected on days 3 and 7 after laser injury, and the expression of miR‐342‐5p and EVL mRNA was compared with the control group. B, Mice underwent laser‐induced CNV and were injected intravitreally with miR‐342‐5p agomir. The level of miR‐342‐5p was determined with quantitative reverse transcription polymerase chain reaction. C, Healthy adult mice underwent laser‐induced CNV . miR‐342‐5p agomir or control was injected intravitreally 24 hours after laser burns. Choroid membranes were collected on day 7 and stained with isolectin B4. The relative CNV areas were compared between the 2 groups. D, A model showing that miR‐342‐5p functions as a multieffect repressor of angiogenesis in EC s. The expression of miR‐342‐5p is downregulated by VEGFR 2 signaling through Akt and upregulated by Notch signaling and TGF ‐β treatment. This mi RNA represses angiogenesis by suppressing VEGFR and endoglin‐mediated TGF ‐β receptor signaling, upregulating Dll4, and downregulating jagged 1 in Notch signaling, leading to decreased EC proliferation, migration, and probably increased endothelial–mesenchymal transition. Bars indicate mean± SD (n=5), * P

    Article Snippet: The γ‐secretase inhibitor (Alexis Biochemicals), VEGF, and TGF‐β (Promega) were used at concentrations of 25, 30, and 2.5 ng/mL, respectively.

    Techniques: Mouse Assay, Expressing, Injection, Reverse Transcription Polymerase Chain Reaction, Staining, Migration

    Oral pretreatment of mice with M. leprae -Hsp65-producing L. lactis was associated with an increased of CD4+LAP+ regulatory T cells expressing TGF-β in mice. C57BL/6 mice were fed or not (Naïve) medium (EAE), control (CT-LL+EAE) or M. leprae -Hsp65-producing L. lactis (Hsp65-LL+EAE) for four days and EAE was induced ten days later. After 1, 2, 4 and 14 days, mice were killed and inguinal (ILN; A) and mesenteric lymph nodes (MLN; B) as well as spleen (SPL; C) removed. Cells were stained with FITC-anti-CD4, PE-anti-CD25, Bio-LAP and CY-STV. Graphs are shown as mean ± SEM. ANOVA, post-test Tukey. *Statistically different from EAE group; p

    Journal: Journal of autoimmunity

    Article Title: Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

    doi: 10.1016/j.jaut.2012.07.012

    Figure Lengend Snippet: Oral pretreatment of mice with M. leprae -Hsp65-producing L. lactis was associated with an increased of CD4+LAP+ regulatory T cells expressing TGF-β in mice. C57BL/6 mice were fed or not (Naïve) medium (EAE), control (CT-LL+EAE) or M. leprae -Hsp65-producing L. lactis (Hsp65-LL+EAE) for four days and EAE was induced ten days later. After 1, 2, 4 and 14 days, mice were killed and inguinal (ILN; A) and mesenteric lymph nodes (MLN; B) as well as spleen (SPL; C) removed. Cells were stained with FITC-anti-CD4, PE-anti-CD25, Bio-LAP and CY-STV. Graphs are shown as mean ± SEM. ANOVA, post-test Tukey. *Statistically different from EAE group; p

    Article Snippet: PE-conjugated mAbs to TGF-β was purchased from IQ Products.

    Techniques: Mouse Assay, Expressing, Staining

    The effects of bradykinin B1 (BKB1) and B2 (BKB2) receptor antagonists on the release of IL-8 ( A ), G-CSF ( B ), MCP-1 ( C ), and TGF-β ( D ) from A549 cell monolayer after a 72-hour incubation ( n = 5). * P

    Journal: The American Journal of Pathology

    Article Title: Bradykinin Stimulates Type II Alveolar Cells to Release Neutrophil and Monocyte Chemotactic Activity and Inflammatory Cytokines

    doi:

    Figure Lengend Snippet: The effects of bradykinin B1 (BKB1) and B2 (BKB2) receptor antagonists on the release of IL-8 ( A ), G-CSF ( B ), MCP-1 ( C ), and TGF-β ( D ) from A549 cell monolayer after a 72-hour incubation ( n = 5). * P

    Article Snippet: The neutralizing antibodies to IL-8, G-CSF, MCP-1, RANTES, GM-CSF, MIP-1α, and TGF-β were purchased from Genzyme (Cambridge, MA) and were added to the A549 cell supernatant fluids, which were harvested at 72 hours in response to 100 μmol/L BK, at the suggested concentration to inhibit these cytokines and incubated for 30 minutes in 37°C before chemotaxis.

    Techniques: Incubation

    The individual time courses of chemokines that comprised chemotactic activity. The release of IL-8 ( A ), G-CSF ( B ), MCP-1 ( C ), and TGF-β ( D ) from A549 cell monolayer in response to 100 μmol/L bradykinin ( n = 6).

    Journal: The American Journal of Pathology

    Article Title: Bradykinin Stimulates Type II Alveolar Cells to Release Neutrophil and Monocyte Chemotactic Activity and Inflammatory Cytokines

    doi:

    Figure Lengend Snippet: The individual time courses of chemokines that comprised chemotactic activity. The release of IL-8 ( A ), G-CSF ( B ), MCP-1 ( C ), and TGF-β ( D ) from A549 cell monolayer in response to 100 μmol/L bradykinin ( n = 6).

    Article Snippet: The neutralizing antibodies to IL-8, G-CSF, MCP-1, RANTES, GM-CSF, MIP-1α, and TGF-β were purchased from Genzyme (Cambridge, MA) and were added to the A549 cell supernatant fluids, which were harvested at 72 hours in response to 100 μmol/L BK, at the suggested concentration to inhibit these cytokines and incubated for 30 minutes in 37°C before chemotaxis.

    Techniques: Activity Assay

    Th17 cell ectonucleotidase expression is driven by tumor-derived TGF-β and IL-6. ( A ) mRNA was extracted from 36 breast cancer tumor samples and the expression of IL17, ENTPD1 and NT5E was determined using RT-qPCR. The correlation between IL17

    Journal: Oncoimmunology

    Article Title: Human ectonucleotidase-expressing CD25high Th17 cells accumulate in breast cancer tumors and exert immunosuppressive functions

    doi: 10.1080/2162402X.2015.1055444

    Figure Lengend Snippet: Th17 cell ectonucleotidase expression is driven by tumor-derived TGF-β and IL-6. ( A ) mRNA was extracted from 36 breast cancer tumor samples and the expression of IL17, ENTPD1 and NT5E was determined using RT-qPCR. The correlation between IL17

    Article Snippet: Human IL-6 (20 ng/mL) and TGF-β (5 ng/mL) were all purchased from MiltenyiBiotec.

    Techniques: Polyacrylamide Gel Electrophoresis, Expressing, Derivative Assay, Quantitative RT-PCR

    Defective expression and activation of TGF-β in mice infected with MHV68-IκBαM. A: Western blot assay for latent form of TGF-β in BAL collected from naïve (N) and MHV68-MR and MHV68-IκBαM–infected

    Journal: The American Journal of Pathology

    Article Title: Inhibition of NF-?B Signaling Reduces Virus Load and Gammaherpesvirus-Induced Pulmonary Fibrosis

    doi: 10.2353/ajpath.2010.091122

    Figure Lengend Snippet: Defective expression and activation of TGF-β in mice infected with MHV68-IκBαM. A: Western blot assay for latent form of TGF-β in BAL collected from naïve (N) and MHV68-MR and MHV68-IκBαM–infected

    Article Snippet: Aliquots of lung lysates (10 μg) and BAL fluid (12 μl) were resolved in 4 to 20% SDS-PAGE, and transferred onto nitrocellulose membranes., , Western blotting for Ym1/2 (kindly provided by Dr. Toshihiko Iwanaga, Hakkaido University Japan), Arginase 1 (Santa Cruz Biotech), Collagen 1 (Santa Cruz biotech), MMP9 (Sigma), and, TGF-β (Pharmingen BD), were performed and filters stripped and re-probed with an antiserum against β-actin (Santa Cruz Biotech) or with an antibody against Surfactant A (Chemicon International) as a loading control for lung homogenates and BAL fluid, respectively.

    Techniques: Expressing, Activation Assay, Mouse Assay, Infection, Western Blot

    Composite profile of Western blot analysis for p16 before and after exposure of proliferating KCs to anti-proliferative reagents (TNF-α, IFN-γ, TGF-β, and TPA) for the indicated time intervals ( A ) and after 48 hours including pretreatment with the MEK inhibitor (PD98059) ( C ). B: Total Ras protein level and activity in KCs before and after exposure to TPA or IFN-γ. D: Western blot analysis of KCs before and after suspension in methylcellulose to detect p12 and p16 levels in the absence and presence of the PKC inhibitor, GF.

    Journal: The American Journal of Pathology

    Article Title: Role of INK4a/Arf Locus-Encoded Senescent Checkpoints Activated in Normal and Psoriatic Keratinocytes

    doi:

    Figure Lengend Snippet: Composite profile of Western blot analysis for p16 before and after exposure of proliferating KCs to anti-proliferative reagents (TNF-α, IFN-γ, TGF-β, and TPA) for the indicated time intervals ( A ) and after 48 hours including pretreatment with the MEK inhibitor (PD98059) ( C ). B: Total Ras protein level and activity in KCs before and after exposure to TPA or IFN-γ. D: Western blot analysis of KCs before and after suspension in methylcellulose to detect p12 and p16 levels in the absence and presence of the PKC inhibitor, GF.

    Article Snippet: Treatments to induce p16 included addition of: recombinant IFN-γ, as well as recombinant TNF-α (103 units/ml; Genentech Inc., San Francisco, CA), TPA (5 nmol/L; Sigma Chemical Co., St. Louis, MO), and TGF-β (10 ng/ml; Upstate Biotechnology, Inc., Lake Placid, NY), in KGM for 24 to 72 hours.

    Techniques: Western Blot, Activity Assay

    PAR‐1 activation induces mesenchymal transition of imPTECs. A, Relative mRNA expression levels of AQP‐1, ZO‐1, vimentin, α‐SMA, fibronectin, and collagen I in imPTECs 24 hours after stimulation with thrombin (1 U/mL) or TGF‐Β (5 ng/mL). Indicated is the average of three independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Protease‐activated receptor‐1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy. Protease‐activated receptor‐1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy

    doi: 10.1111/jcmm.14028

    Figure Lengend Snippet: PAR‐1 activation induces mesenchymal transition of imPTECs. A, Relative mRNA expression levels of AQP‐1, ZO‐1, vimentin, α‐SMA, fibronectin, and collagen I in imPTECs 24 hours after stimulation with thrombin (1 U/mL) or TGF‐Β (5 ng/mL). Indicated is the average of three independent experiments. * P

    Article Snippet: Cells were starved at least 4 hours prior to stimulation with 1 U/mL thrombin (Sigma, St. Louis, Missouri, USA), 100 μM PAR‐1 agonist peptide (TRAP6; H‐SFLLRN‐NH2; Biochem, Shanghai, China), or 5 μg/mL TGF‐β (Tebu‐bio, Heerhugowaard, the Netherlands).

    Techniques: Activation Assay, Expressing

    PAR‐1 activation induces pro‐fibrotic cytokine expression and potentiates macrophage influx during renal fibrosis. A, imPTEC mRNA expression of cytokines 24 h after PAR‐1 stimulation with thrombin (1 U/mL). Indicated is the average of three independent experiments. B‐C, Protein expression of MCP‐1 (B), total TGF‐β and active TGF‐β (C) measured by ELISA in whole kidney lysates of wild‐type and PAR‐1‐deficient (PAR‐1 −/− ) mice 7 and 10 d after UUO and in unobstructed control kidneys. D, F4/80 (ie, macrophage marker) mRNA expression in kidney lysates of wild‐type and PAR‐1‐deficient (PAR‐1 −/− ) mice 1, 3, 7, and 10 d after UUO and in unobstructed (Sham) control kidneys. E: Representative images of F4/80‐stained kidney slides of wild‐type and PAR‐1‐deficient (PAR‐1 −/− ) mice 1, 3, 7, and 10 d after UUO and in unobstructed (Sham) control kidneys; scale bars represent 100 μm. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Protease‐activated receptor‐1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy. Protease‐activated receptor‐1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy

    doi: 10.1111/jcmm.14028

    Figure Lengend Snippet: PAR‐1 activation induces pro‐fibrotic cytokine expression and potentiates macrophage influx during renal fibrosis. A, imPTEC mRNA expression of cytokines 24 h after PAR‐1 stimulation with thrombin (1 U/mL). Indicated is the average of three independent experiments. B‐C, Protein expression of MCP‐1 (B), total TGF‐β and active TGF‐β (C) measured by ELISA in whole kidney lysates of wild‐type and PAR‐1‐deficient (PAR‐1 −/− ) mice 7 and 10 d after UUO and in unobstructed control kidneys. D, F4/80 (ie, macrophage marker) mRNA expression in kidney lysates of wild‐type and PAR‐1‐deficient (PAR‐1 −/− ) mice 1, 3, 7, and 10 d after UUO and in unobstructed (Sham) control kidneys. E: Representative images of F4/80‐stained kidney slides of wild‐type and PAR‐1‐deficient (PAR‐1 −/− ) mice 1, 3, 7, and 10 d after UUO and in unobstructed (Sham) control kidneys; scale bars represent 100 μm. * P

    Article Snippet: Cells were starved at least 4 hours prior to stimulation with 1 U/mL thrombin (Sigma, St. Louis, Missouri, USA), 100 μM PAR‐1 agonist peptide (TRAP6; H‐SFLLRN‐NH2; Biochem, Shanghai, China), or 5 μg/mL TGF‐β (Tebu‐bio, Heerhugowaard, the Netherlands).

    Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Mouse Assay, Marker, Staining

    Treatment with IL-2C increased the expression of M2-associated markers in the contusion margin. (A) Co-localization of CD206 with the microglia marker Iba1 showed that IL-2C increased the number of M2 microglia on day 3 post-injury in the contusion area. (B) Cell count analyses indicated that IL-2C significantly increased the amount of CD206 and Iba-1-double-positive cells. (C,D) The traumatic brain injury TBI-induced expression of TGF-β in the ipsilateral cortices on days 1 and 3 post-injury was increased by treatment with IL-2C. (E,F) Immunohistochemistry showed that treatment with IL-2C significantly increased the number of arginase-1 + cells around the lesion area. Data are presented as the mean ± SD; * p

    Journal: Frontiers in Neurology

    Article Title: IL-2/Anti-IL-2 Complex Attenuates Inflammation and BBB Disruption in Mice Subjected to Traumatic Brain Injury

    doi: 10.3389/fneur.2017.00281

    Figure Lengend Snippet: Treatment with IL-2C increased the expression of M2-associated markers in the contusion margin. (A) Co-localization of CD206 with the microglia marker Iba1 showed that IL-2C increased the number of M2 microglia on day 3 post-injury in the contusion area. (B) Cell count analyses indicated that IL-2C significantly increased the amount of CD206 and Iba-1-double-positive cells. (C,D) The traumatic brain injury TBI-induced expression of TGF-β in the ipsilateral cortices on days 1 and 3 post-injury was increased by treatment with IL-2C. (E,F) Immunohistochemistry showed that treatment with IL-2C significantly increased the number of arginase-1 + cells around the lesion area. Data are presented as the mean ± SD; * p

    Article Snippet: The relevant proteins were detected by incubating the membranes with primary antibodies overnight (i.e., β-actin, 1:1,000, CST, Cat #: 4970S; TNF-α, 1:10,000, GeneTex, Cat #: GTX110520; IL-1β, 1:1,000, CST, Cat #: 12242S; TLR4, 1:1,000, Abcam, Cat #: ab13556; NF-κB, 1:1,000, CST, Cat #: 8482S; TGF-β, 1:2,000, Torrey Pines Biolabs; ZO-1, 1:1,000, Invitrogen, Cat #: 40-2200; and occludin, 1:1,000, Invitrogen, Cat #: 33-1500) and then with secondary antibodies (i.e., horseradish-peroxidase conjugated goat anti-rabbit or anti-mouse IgG, 1:3,000, Cell Signaling Technology).

    Techniques: Expressing, Marker, Cell Counting, Immunohistochemistry

    Expression levels of cytokines (A) IL-6, (B) IL-10, (C) IL-23 and (D) TGF-β, as determined by ELISA. The horizontal line represents the mean values for each group. IL, interleukin; TGF, transforming growth factor.

    Journal: Experimental and Therapeutic Medicine

    Article Title: FOXP3+ associated with the pro-inflammatory regulatory T and T helper 17 effector cells in asthma patients

    doi: 10.3892/etm.2016.3662

    Figure Lengend Snippet: Expression levels of cytokines (A) IL-6, (B) IL-10, (C) IL-23 and (D) TGF-β, as determined by ELISA. The horizontal line represents the mean values for each group. IL, interleukin; TGF, transforming growth factor.

    Article Snippet: Expression levels of IL-6 (EH2IL62), IL-23 (KHC0231; both Thermo Fisher Scientific, Inc.), IL-10 (RAB1060; Sigma-Aldrich; Merck Millipore)and TGF-β (E-EL-H0110c; Elabscience Bioengineering Co., Ltd., Wuhan, China) were detected in BALF samples obtained from the patients and control subjects using respective ELISA kits according to the manufacturer's protocols.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    a Effect of VCN-2 hydrocolloid film on expression of mediators after 14 days treatment was detected by Western blotting. Down-regulation of various anti-inflammatory and up-regulation of expression of several wound healing markers in skin tissues were observed. β-actin acted as control marker. b Effect of VCN-2 film on expression of mediators after 14 days topically treatment was detected by immunoblotting. A graph represented densitometry analysis results of the effect of VCN-2 on proteins expression proinflammatory mediators ( c ) anti-HIF1α, ( d ) MMP-9, and ( e ) VEGF and TGF-β. All data are expressed as mean ± SEM. ### p

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Improvement of diabetic wound healing by topical application of Vicenin-2 hydrocolloid film on Sprague Dawley rats

    doi: 10.1186/s12906-018-2427-y

    Figure Lengend Snippet: a Effect of VCN-2 hydrocolloid film on expression of mediators after 14 days treatment was detected by Western blotting. Down-regulation of various anti-inflammatory and up-regulation of expression of several wound healing markers in skin tissues were observed. β-actin acted as control marker. b Effect of VCN-2 film on expression of mediators after 14 days topically treatment was detected by immunoblotting. A graph represented densitometry analysis results of the effect of VCN-2 on proteins expression proinflammatory mediators ( c ) anti-HIF1α, ( d ) MMP-9, and ( e ) VEGF and TGF-β. All data are expressed as mean ± SEM. ### p

    Article Snippet: Diagnostic kits for VEGF and TGF-β were bought from Bender MedSystems (Vienna, Austria) while insulin diagnostic kits were acquired from Mercodia Corporation (Uppsala, Sweden).

    Techniques: Expressing, Western Blot, Marker

    GD2-CAR-engineered T cells, expanded by the allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP), are more resistant to immunosuppressive cytokines and oxidative and apoptotic stress than GD2-CAR T cells expanded by the rapid expansion protocol (REP). ( a ) An illustration of the GD2-CAR-encoding lentiviral vector used to transduce T cells. ( b ) CD8 + T cells were transduced, isolated by magnetic beads, and expanded by the REP or AEP protocols. The starting number of GD2-CAR T cells was 1 × 10 5 for both protocols and a ratio of 1:1:4 (T-cell:mDC:ASAL) was used in the AEP protocol. Fold expansion of GD2-CAR T cells is presented. ( c ) The cytotoxic ability of GD2-CAR T cells was analyzed by coculture with luciferase-expressing GD2 + (IMR-32) or GD2 − (SK-N-FI) neuroblastoma target cells at different E:T ratios. Viability of target cells were measured by luminescence and related to signals from target cells alone. ( d ) GD2-CAR T cells expanded by the REP and AEP protocols were exposed to either 25 μmol/l H 2 O 2 or 0.1 μmol/l doxorubicin for 24 hours. Viability was analyzed by flow cytometry using Annexin-V and PI staining and relative viability was normalized against nontreated GD2-CAR T cells. ( e–f ) The REP- and AEP-expanded GD2-CAR T cells were treated with IL-10/TGF-β for 4 hours or with H 2 O 2 for 24 hours, followed by 4 hours treatment with IL-10/TGF-β and then cocultured with GD2 + target cells (IMR-32) in the presence of IL-10/TGF-β for 24 hours. ( e ) Flow cytometry was used to analyze CD107a expression on GD2-CAR T cells (CD3 + ) after exposure to IL-10/TGF-β or H 2 O 2 /IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. ( f ) ELISA was used to analyze IFN-γ production from GD2-CAR T cells after exposure to IL-10/TGF-β or H 2 O 2 /IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. ( g ) GD2-CAR T cells were treated with H 2 O 2 for 24 hours, followed by 4 hours labeling with CFSE in the presence of IL-10/TGF-β and then cocultured with GD2 + target cells (IMR-32) in the presence of IL-10/TGF-β for 4 days before proliferation analysis by flow cytometry. ( a–g ) The experiments were performed three times with three new and different donors each time. Error bars represent SD, and statistical significance is depicted by symbols (* P

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors

    doi: 10.1038/mtm.2014.1

    Figure Lengend Snippet: GD2-CAR-engineered T cells, expanded by the allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP), are more resistant to immunosuppressive cytokines and oxidative and apoptotic stress than GD2-CAR T cells expanded by the rapid expansion protocol (REP). ( a ) An illustration of the GD2-CAR-encoding lentiviral vector used to transduce T cells. ( b ) CD8 + T cells were transduced, isolated by magnetic beads, and expanded by the REP or AEP protocols. The starting number of GD2-CAR T cells was 1 × 10 5 for both protocols and a ratio of 1:1:4 (T-cell:mDC:ASAL) was used in the AEP protocol. Fold expansion of GD2-CAR T cells is presented. ( c ) The cytotoxic ability of GD2-CAR T cells was analyzed by coculture with luciferase-expressing GD2 + (IMR-32) or GD2 − (SK-N-FI) neuroblastoma target cells at different E:T ratios. Viability of target cells were measured by luminescence and related to signals from target cells alone. ( d ) GD2-CAR T cells expanded by the REP and AEP protocols were exposed to either 25 μmol/l H 2 O 2 or 0.1 μmol/l doxorubicin for 24 hours. Viability was analyzed by flow cytometry using Annexin-V and PI staining and relative viability was normalized against nontreated GD2-CAR T cells. ( e–f ) The REP- and AEP-expanded GD2-CAR T cells were treated with IL-10/TGF-β for 4 hours or with H 2 O 2 for 24 hours, followed by 4 hours treatment with IL-10/TGF-β and then cocultured with GD2 + target cells (IMR-32) in the presence of IL-10/TGF-β for 24 hours. ( e ) Flow cytometry was used to analyze CD107a expression on GD2-CAR T cells (CD3 + ) after exposure to IL-10/TGF-β or H 2 O 2 /IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. ( f ) ELISA was used to analyze IFN-γ production from GD2-CAR T cells after exposure to IL-10/TGF-β or H 2 O 2 /IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. ( g ) GD2-CAR T cells were treated with H 2 O 2 for 24 hours, followed by 4 hours labeling with CFSE in the presence of IL-10/TGF-β and then cocultured with GD2 + target cells (IMR-32) in the presence of IL-10/TGF-β for 4 days before proliferation analysis by flow cytometry. ( a–g ) The experiments were performed three times with three new and different donors each time. Error bars represent SD, and statistical significance is depicted by symbols (* P

    Article Snippet: They were then treated with H2 O2 for 24 hours, washed twice, incubated with IL-10 (Nordic Biosite, 2.5 ng/ml) and TGF-β (Nordic Biosite, 10 ng/ml) for 4 hours and then mixed with target cells at an effector:target cell (E:T) ratio of 1:1.

    Techniques: Plasmid Preparation, Transduction, Isolation, Magnetic Beads, Luciferase, Expressing, Flow Cytometry, Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Labeling