glucagon purification recombinant glucagon Search Results


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  • 99
    Thermo Fisher topo ta cloning kit
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glp 1
    Serum levels of glucose, insulin, and <t>GLP-1</t> after Ad-GLP-1 transduction. Two groups of mice were transduced (n = 10/group) and subjected to an overnight fast. Sera were obtained from five mice per group and assayed for GLP-1(7–36-amide)
    Glp 1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human hgf
    Cdk2- and cyclin E-associated kinase activities of <t>TGF-β-</t> and <t>HGF-treated</t> Mv1Lu cells. Cells were treated with the indicated growth factors, followed by lysis in NP-40 buffer. Immunoprecipitations with the indicated antibodies and kinase assays were carried out as described in Materials and Methods. (A) Cdk2-associated kinase activity with histone H1 as a substrate. (B) Cdk2 (lanes 1 to 4)- and cyclin E (lanes 5 to 8)-associated kinase activities with GST-Rb as a substrate. The figures show relative kinase activities compared to control cells (set at 100). (C) Sparsely seeded cells were pretreated with TGF-β (400 pM) for 16 h, followed by addition of HGF (220 pM) without removal of TGF-β, and incubations were continued for the indicated times (4 to 36 h). Untreated control cells are included (lanes 1 and 14). In wash , cells treated with TGF-β for 16 h were washed four times with growth medium, followed by addition of fresh medium. The cells were lysed, followed by Cdk6 and Cdk2 immunoblotting and determination of Cdk2-associated kinase activity towards histone H1. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum are shown compared to cells treated with TGF-β for 16 h (set at 100) (lane 1). 5-BrdUrd incorporation was analyzed for the last hour of each incubation. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei.
    Recombinant Human Hgf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 97/100, based on 475 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cedarlane anti mouse human mac 2 galectin 3 purified clone m3 38 rat igg2a
    Cdk2- and cyclin E-associated kinase activities of <t>TGF-β-</t> and <t>HGF-treated</t> Mv1Lu cells. Cells were treated with the indicated growth factors, followed by lysis in NP-40 buffer. Immunoprecipitations with the indicated antibodies and kinase assays were carried out as described in Materials and Methods. (A) Cdk2-associated kinase activity with histone H1 as a substrate. (B) Cdk2 (lanes 1 to 4)- and cyclin E (lanes 5 to 8)-associated kinase activities with GST-Rb as a substrate. The figures show relative kinase activities compared to control cells (set at 100). (C) Sparsely seeded cells were pretreated with TGF-β (400 pM) for 16 h, followed by addition of HGF (220 pM) without removal of TGF-β, and incubations were continued for the indicated times (4 to 36 h). Untreated control cells are included (lanes 1 and 14). In wash , cells treated with TGF-β for 16 h were washed four times with growth medium, followed by addition of fresh medium. The cells were lysed, followed by Cdk6 and Cdk2 immunoblotting and determination of Cdk2-associated kinase activity towards histone H1. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum are shown compared to cells treated with TGF-β for 16 h (set at 100) (lane 1). 5-BrdUrd incorporation was analyzed for the last hour of each incubation. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei.
    Anti Mouse Human Mac 2 Galectin 3 Purified Clone M3 38 Rat Igg2a, supplied by Cedarlane, used in various techniques. Bioz Stars score: 99/100, based on 622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti sp1
    The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, <t>Sp1,</t> p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P
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    Bethyl rabbit anti mafa ihc antibody affinity purified
    The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, <t>Sp1,</t> p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P
    Rabbit Anti Mafa Ihc Antibody Affinity Purified, supplied by Bethyl, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant hgf
    The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, <t>Sp1,</t> p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P
    Recombinant Hgf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human pdx 1 ipf1 antibody
    The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, <t>Sp1,</t> p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P
    Human Pdx 1 Ipf1 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher l glutamine
    The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, <t>Sp1,</t> p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P
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    Cell Signaling Technology Inc tumor necrosis factor tnf α
    Proposed mechanisms underlying the positive therapeutic effects of combined melatonin and exendin-4 therapy on reversing the deterioration of kidney function from cardiorenal syndrome (CRS). LEVF = left ventricular ejection fraction; <t>TNF-α</t> = tumor necrosis factor alpha; NF-κB = nuclear factor-κB; MMP-9 = matrix metalloproteinase 9; iNOS = inducible nitric oxide synthase; HO-1 = heme oxygenase 1; TGF-β = transforming growth factor beta; c-PARP = cleaved Poly (ADP-ribose) polymerase.
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    R&D Systems mouse hgf
    Proposed mechanisms underlying the positive therapeutic effects of combined melatonin and exendin-4 therapy on reversing the deterioration of kidney function from cardiorenal syndrome (CRS). LEVF = left ventricular ejection fraction; <t>TNF-α</t> = tumor necrosis factor alpha; NF-κB = nuclear factor-κB; MMP-9 = matrix metalloproteinase 9; iNOS = inducible nitric oxide synthase; HO-1 = heme oxygenase 1; TGF-β = transforming growth factor beta; c-PARP = cleaved Poly (ADP-ribose) polymerase.
    Mouse Hgf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher accuprime pfx dna polymerase
    Proposed mechanisms underlying the positive therapeutic effects of combined melatonin and exendin-4 therapy on reversing the deterioration of kidney function from cardiorenal syndrome (CRS). LEVF = left ventricular ejection fraction; <t>TNF-α</t> = tumor necrosis factor alpha; NF-κB = nuclear factor-κB; MMP-9 = matrix metalloproteinase 9; iNOS = inducible nitric oxide synthase; HO-1 = heme oxygenase 1; TGF-β = transforming growth factor beta; c-PARP = cleaved Poly (ADP-ribose) polymerase.
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    Thermo Fisher alexa 594 conjugated donkey antibodies
    Proposed mechanisms underlying the positive therapeutic effects of combined melatonin and exendin-4 therapy on reversing the deterioration of kidney function from cardiorenal syndrome (CRS). LEVF = left ventricular ejection fraction; <t>TNF-α</t> = tumor necrosis factor alpha; NF-κB = nuclear factor-κB; MMP-9 = matrix metalloproteinase 9; iNOS = inducible nitric oxide synthase; HO-1 = heme oxygenase 1; TGF-β = transforming growth factor beta; c-PARP = cleaved Poly (ADP-ribose) polymerase.
    Alexa 594 Conjugated Donkey Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor
    Proposed mechanisms underlying the positive therapeutic effects of combined melatonin and exendin-4 therapy on reversing the deterioration of kidney function from cardiorenal syndrome (CRS). LEVF = left ventricular ejection fraction; <t>TNF-α</t> = tumor necrosis factor alpha; NF-κB = nuclear factor-κB; MMP-9 = matrix metalloproteinase 9; iNOS = inducible nitric oxide synthase; HO-1 = heme oxygenase 1; TGF-β = transforming growth factor beta; c-PARP = cleaved Poly (ADP-ribose) polymerase.
    Alexa Fluor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibodies against foxo1
    <t>FoxO1-S253</t> A/A mice exhibit impaired glucagon tolerance test and hepatic gene expression. (A) Glycogen concentration in the liver of mice at 16-hour fasting and random-fed states. * P
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    Cell Signaling Technology Inc anti akt
    <t>FoxO1-S253</t> A/A mice exhibit impaired glucagon tolerance test and hepatic gene expression. (A) Glycogen concentration in the liver of mice at 16-hour fasting and random-fed states. * P
    Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 17252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti as160
    Action of conditioned medium on Akt, ERK, <t>AS160,</t> and Akt substrates phosphorylation after glucose stimulation, or IRS-1 and -2 protein expression in rat primary β-cells. Conditioned media and abbreviations as described in the legend to Fig. 3 . After 24-h culture in conditioned medium, β-cells were incubated 1 h at 2.8 mmol/L or 16.7 mmol/L glucose (Glc). Open bars = 2.8 mmol/L glucose; closed bars = 16.7 mmol/L glucose. Western blots were scanned and data normalized to total protein or actin as indicated. N = 3 independent experiments. A : Akt Ser 473 phosphorylation. * P
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    Cell Signaling Technology Inc anti erk
    Action of conditioned medium on Akt, ERK, <t>AS160,</t> and Akt substrates phosphorylation after glucose stimulation, or IRS-1 and -2 protein expression in rat primary β-cells. Conditioned media and abbreviations as described in the legend to Fig. 3 . After 24-h culture in conditioned medium, β-cells were incubated 1 h at 2.8 mmol/L or 16.7 mmol/L glucose (Glc). Open bars = 2.8 mmol/L glucose; closed bars = 16.7 mmol/L glucose. Western blots were scanned and data normalized to total protein or actin as indicated. N = 3 independent experiments. A : Akt Ser 473 phosphorylation. * P
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    Millipore anti fibronectin antibody
    Establishment of a murine breast cancer cell line undergoing TGFβ-induced EMT. ( A ) Primary tumor cells were isolated from an advanced breast tumor of a MMTV-PyMT transgenic female mouse and were cultured for at least 2 months prior to further experimentation, resulting in a novel cell line termed Py2T. ( B ) Py2T cells maintain the MMTV-PyMT transgene. The MMTV-PyMT transgene was detected by PCR and agarose gel electrophoresis. DNA from an MMTV-PyMT tumor and from normal murine mammary gland (NMuMG) cells served as positive and negative controls, respectively. ( C ) Py2T cells lost the expression of the MMTV-PyMT transgene. Immunoblotting for the PyMT protein was performed on lysates of Py2T cells untreated or treated with 0.1 µM Dexamethasone for up to 72 h to induce the MMTV promoter. Lysates of an MMTV-PyMT tumor and NMuMG cells served as positive and negative controls, respectively. ( D ) Treatment of Py2T cells with known EMT inducers. Cells were continuously treated with the indicated growth factors and cytokines for 10 days (2 ng/mL TGFβ1; 50 ng/mL EGF; 10 ng/mL IGF-I; 50 ng/mL HGF; 20 ng/mL FGF-2; 20 ng/mL PDGF-BB; 50 ng/mL IL-6). Potential morphological changes were analyzed by phase-contrast microscopy. ( E ) Expression of epithelial (E-cadherin) and mesenchymal (N-cadherin, <t>fibronectin)</t> markers were analyzed by immunoblotting of the lysates of cells treated in (D). ( F ) Immunoblotting analysis of EMT marker expression in Py2T and Py2T LT cells. The mesenchymal subline Py2T LT (long-term) was generated by TGFβ-treatment of Py2T cells for at least 20 days, and was subsequently maintained in TGFβ containing growth medium. ( G ) Analysis of markers for EMT and breast cell type before and after TGFβ-induced EMT. Immunofluorescence staining was performed with antibodies against E-Cadherin (epithelial marker), vimentin (mesenchymal marker), estrogen receptor alpha (ERα), cytokeratin 8/18 (luminal markers) and cytokeratin 14 (basal marker). Scale bar, 20 µm.
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    Establishment of a murine breast cancer cell line undergoing TGFβ-induced EMT. ( A ) Primary tumor cells were isolated from an advanced breast tumor of a MMTV-PyMT transgenic female mouse and were cultured for at least 2 months prior to further experimentation, resulting in a novel cell line termed Py2T. ( B ) Py2T cells maintain the MMTV-PyMT transgene. The MMTV-PyMT transgene was detected by PCR and agarose gel electrophoresis. DNA from an MMTV-PyMT tumor and from normal murine mammary gland (NMuMG) cells served as positive and negative controls, respectively. ( C ) Py2T cells lost the expression of the MMTV-PyMT transgene. Immunoblotting for the PyMT protein was performed on lysates of Py2T cells untreated or treated with 0.1 µM Dexamethasone for up to 72 h to induce the MMTV promoter. Lysates of an MMTV-PyMT tumor and NMuMG cells served as positive and negative controls, respectively. ( D ) Treatment of Py2T cells with known EMT inducers. Cells were continuously treated with the indicated growth factors and cytokines for 10 days (2 ng/mL TGFβ1; 50 ng/mL EGF; 10 ng/mL IGF-I; 50 ng/mL HGF; 20 ng/mL FGF-2; 20 ng/mL PDGF-BB; 50 ng/mL IL-6). Potential morphological changes were analyzed by phase-contrast microscopy. ( E ) Expression of epithelial (E-cadherin) and mesenchymal (N-cadherin, <t>fibronectin)</t> markers were analyzed by immunoblotting of the lysates of cells treated in (D). ( F ) Immunoblotting analysis of EMT marker expression in Py2T and Py2T LT cells. The mesenchymal subline Py2T LT (long-term) was generated by TGFβ-treatment of Py2T cells for at least 20 days, and was subsequently maintained in TGFβ containing growth medium. ( G ) Analysis of markers for EMT and breast cell type before and after TGFβ-induced EMT. Immunofluorescence staining was performed with antibodies against E-Cadherin (epithelial marker), vimentin (mesenchymal marker), estrogen receptor alpha (ERα), cytokeratin 8/18 (luminal markers) and cytokeratin 14 (basal marker). Scale bar, 20 µm.
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    Identification of HMGB1 as a novel <t>metformin-binding</t> protein. A , structure of metformin ( upper ) and the biotinylated compound containing metformin-like biguanide structure ( lower ) used for the affinity chromatography. B , affinity purification was performed from rat liver cytosol with the metformin beads and the control biotin beads. Bead eluates were analyzed by SDS-PAGE followed by silver staining. Mass spectrometry analysis revealed the ∼25-kDa protein indicated by the arrow was HMGB1. C , HMGB1 in cytosolic fraction of HepG2 cells was pulled down with the metformin beads in the presence of indicated concentrations of metformin. D , recombinant HMGB1 was pulled down with the metformin beads or control beads in the presence of the indicated concentrations of metformin, phenformin, putrescine, or 6-aminohexanoic acid. Data shown are representative of three independent experiments with similar results.
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    Identification of HMGB1 as a novel <t>metformin-binding</t> protein. A , structure of metformin ( upper ) and the biotinylated compound containing metformin-like biguanide structure ( lower ) used for the affinity chromatography. B , affinity purification was performed from rat liver cytosol with the metformin beads and the control biotin beads. Bead eluates were analyzed by SDS-PAGE followed by silver staining. Mass spectrometry analysis revealed the ∼25-kDa protein indicated by the arrow was HMGB1. C , HMGB1 in cytosolic fraction of HepG2 cells was pulled down with the metformin beads in the presence of indicated concentrations of metformin. D , recombinant HMGB1 was pulled down with the metformin beads or control beads in the presence of the indicated concentrations of metformin, phenformin, putrescine, or 6-aminohexanoic acid. Data shown are representative of three independent experiments with similar results.
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    Identification of HMGB1 as a novel <t>metformin-binding</t> protein. A , structure of metformin ( upper ) and the biotinylated compound containing metformin-like biguanide structure ( lower ) used for the affinity chromatography. B , affinity purification was performed from rat liver cytosol with the metformin beads and the control biotin beads. Bead eluates were analyzed by SDS-PAGE followed by silver staining. Mass spectrometry analysis revealed the ∼25-kDa protein indicated by the arrow was HMGB1. C , HMGB1 in cytosolic fraction of HepG2 cells was pulled down with the metformin beads in the presence of indicated concentrations of metformin. D , recombinant HMGB1 was pulled down with the metformin beads or control beads in the presence of the indicated concentrations of metformin, phenformin, putrescine, or 6-aminohexanoic acid. Data shown are representative of three independent experiments with similar results.
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    Identification of HMGB1 as a novel <t>metformin-binding</t> protein. A , structure of metformin ( upper ) and the biotinylated compound containing metformin-like biguanide structure ( lower ) used for the affinity chromatography. B , affinity purification was performed from rat liver cytosol with the metformin beads and the control biotin beads. Bead eluates were analyzed by SDS-PAGE followed by silver staining. Mass spectrometry analysis revealed the ∼25-kDa protein indicated by the arrow was HMGB1. C , HMGB1 in cytosolic fraction of HepG2 cells was pulled down with the metformin beads in the presence of indicated concentrations of metformin. D , recombinant HMGB1 was pulled down with the metformin beads or control beads in the presence of the indicated concentrations of metformin, phenformin, putrescine, or 6-aminohexanoic acid. Data shown are representative of three independent experiments with similar results.
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    Site-specific incorporation of AzF into recombinant <t>GLP-1</t> fused to sfGFP. ( A ) Coomassie Brilliant Blue-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) image of cell lysates before (BI) and after (AI) induction; the prominent band attributable to the sfGFP-GLP-1 fusion protein is indicated (arrow). Protein molecular weight standards are shown in lane M. ( B ) Purified sfGFP-GLP1_16AzF on Coomassie Brilliant Blue-stained SDS-PAGE gel after nickel–nitrilotriacetic acid (Ni-NTA) chromatography and anion exchange chromatography (arrow). Protein molecular weight standards are shown in lane M. ( C ) Protein gel image of sfGFP-GLP1_16AzF treated with or without fluorescent dye (DBCO-PEG4-carboxyrhodamine). The gel was subjected to UV irradiation (302 nm) to excite the fluorophore (fluorescence) and stained with Coomassie Brilliant Blue (Coomassie) to visualize the protein.
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    Image Search Results


    Serum levels of glucose, insulin, and GLP-1 after Ad-GLP-1 transduction. Two groups of mice were transduced (n = 10/group) and subjected to an overnight fast. Sera were obtained from five mice per group and assayed for GLP-1(7–36-amide)

    Journal: Endocrinology

    Article Title: Systemic Delivery of Bioactive Glucagon-Like Peptide 1 after Adenoviral-Mediated Gene Transfer in the Murine Salivary Gland

    doi: 10.1210/en.2010-0193

    Figure Lengend Snippet: Serum levels of glucose, insulin, and GLP-1 after Ad-GLP-1 transduction. Two groups of mice were transduced (n = 10/group) and subjected to an overnight fast. Sera were obtained from five mice per group and assayed for GLP-1(7–36-amide)

    Article Snippet: To demonstrate that our transgenic GLP-1 was bioactive, we incubated the COS7 conditioned medium containing the transgenic GLP-1 (7–37) with NIT1 cells, a mouse pancreatic β-cell line ( ) that secretes RSP granule cargo including insulin in response to GLP-1 ( ).

    Techniques: Transduction, Mouse Assay

    A, Sequence of the engineered GLP-1 gene. The Ala to Gly substitution is highlighted in green . B, Expression and secretion of GLP-1 [GLP-1 (7–37) and/or GLP-1(7–36-amide)] after transfection of the plasmid in Neuro2a cells.

    Journal: Endocrinology

    Article Title: Systemic Delivery of Bioactive Glucagon-Like Peptide 1 after Adenoviral-Mediated Gene Transfer in the Murine Salivary Gland

    doi: 10.1210/en.2010-0193

    Figure Lengend Snippet: A, Sequence of the engineered GLP-1 gene. The Ala to Gly substitution is highlighted in green . B, Expression and secretion of GLP-1 [GLP-1 (7–37) and/or GLP-1(7–36-amide)] after transfection of the plasmid in Neuro2a cells.

    Article Snippet: To demonstrate that our transgenic GLP-1 was bioactive, we incubated the COS7 conditioned medium containing the transgenic GLP-1 (7–37) with NIT1 cells, a mouse pancreatic β-cell line ( ) that secretes RSP granule cargo including insulin in response to GLP-1 ( ).

    Techniques: Sequencing, Expressing, Transfection, Plasmid Preparation

    GTTs in mice before (A) and after transduction with either Ad-GLP-1 or Ad-Luc. Note that the clearance rate of glucose for mice treated with the control vector is not different before or after vector administration (B), whereas the mice administered the

    Journal: Endocrinology

    Article Title: Systemic Delivery of Bioactive Glucagon-Like Peptide 1 after Adenoviral-Mediated Gene Transfer in the Murine Salivary Gland

    doi: 10.1210/en.2010-0193

    Figure Lengend Snippet: GTTs in mice before (A) and after transduction with either Ad-GLP-1 or Ad-Luc. Note that the clearance rate of glucose for mice treated with the control vector is not different before or after vector administration (B), whereas the mice administered the

    Article Snippet: To demonstrate that our transgenic GLP-1 was bioactive, we incubated the COS7 conditioned medium containing the transgenic GLP-1 (7–37) with NIT1 cells, a mouse pancreatic β-cell line ( ) that secretes RSP granule cargo including insulin in response to GLP-1 ( ).

    Techniques: Mouse Assay, Transduction, Plasmid Preparation

    A, Scatter plot of blood glucose levels from mice treated with alloxan and Ad-GLP-1 or Ad-Luc. Note that both groups of animals developed diabetes in a time-dependent manner; however, the group of Ad-GLP-1 transduced mice ( filled squares ) were significantly

    Journal: Endocrinology

    Article Title: Systemic Delivery of Bioactive Glucagon-Like Peptide 1 after Adenoviral-Mediated Gene Transfer in the Murine Salivary Gland

    doi: 10.1210/en.2010-0193

    Figure Lengend Snippet: A, Scatter plot of blood glucose levels from mice treated with alloxan and Ad-GLP-1 or Ad-Luc. Note that both groups of animals developed diabetes in a time-dependent manner; however, the group of Ad-GLP-1 transduced mice ( filled squares ) were significantly

    Article Snippet: To demonstrate that our transgenic GLP-1 was bioactive, we incubated the COS7 conditioned medium containing the transgenic GLP-1 (7–37) with NIT1 cells, a mouse pancreatic β-cell line ( ) that secretes RSP granule cargo including insulin in response to GLP-1 ( ).

    Techniques: Mouse Assay

    Cdk2- and cyclin E-associated kinase activities of TGF-β- and HGF-treated Mv1Lu cells. Cells were treated with the indicated growth factors, followed by lysis in NP-40 buffer. Immunoprecipitations with the indicated antibodies and kinase assays were carried out as described in Materials and Methods. (A) Cdk2-associated kinase activity with histone H1 as a substrate. (B) Cdk2 (lanes 1 to 4)- and cyclin E (lanes 5 to 8)-associated kinase activities with GST-Rb as a substrate. The figures show relative kinase activities compared to control cells (set at 100). (C) Sparsely seeded cells were pretreated with TGF-β (400 pM) for 16 h, followed by addition of HGF (220 pM) without removal of TGF-β, and incubations were continued for the indicated times (4 to 36 h). Untreated control cells are included (lanes 1 and 14). In wash , cells treated with TGF-β for 16 h were washed four times with growth medium, followed by addition of fresh medium. The cells were lysed, followed by Cdk6 and Cdk2 immunoblotting and determination of Cdk2-associated kinase activity towards histone H1. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum are shown compared to cells treated with TGF-β for 16 h (set at 100) (lane 1). 5-BrdUrd incorporation was analyzed for the last hour of each incubation. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei.

    Journal: Molecular and Cellular Biology

    Article Title: Hepatocyte Growth Factor Releases Mink Epithelial Cells from Transforming Growth Factor ?1-Induced Growth Arrest by Restoring Cdk6 Expression and Cyclin E-Associated Cdk2 Activity

    doi:

    Figure Lengend Snippet: Cdk2- and cyclin E-associated kinase activities of TGF-β- and HGF-treated Mv1Lu cells. Cells were treated with the indicated growth factors, followed by lysis in NP-40 buffer. Immunoprecipitations with the indicated antibodies and kinase assays were carried out as described in Materials and Methods. (A) Cdk2-associated kinase activity with histone H1 as a substrate. (B) Cdk2 (lanes 1 to 4)- and cyclin E (lanes 5 to 8)-associated kinase activities with GST-Rb as a substrate. The figures show relative kinase activities compared to control cells (set at 100). (C) Sparsely seeded cells were pretreated with TGF-β (400 pM) for 16 h, followed by addition of HGF (220 pM) without removal of TGF-β, and incubations were continued for the indicated times (4 to 36 h). Untreated control cells are included (lanes 1 and 14). In wash , cells treated with TGF-β for 16 h were washed four times with growth medium, followed by addition of fresh medium. The cells were lysed, followed by Cdk6 and Cdk2 immunoblotting and determination of Cdk2-associated kinase activity towards histone H1. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum are shown compared to cells treated with TGF-β for 16 h (set at 100) (lane 1). 5-BrdUrd incorporation was analyzed for the last hour of each incubation. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei.

    Article Snippet: In all assays, except when otherwise stated, the cells were treated with 100 pM TGF-β1, 220 pM HGF, or both in DMEM containing 10% FCS for 16 h. TGF-β1 was purified from outdated human platelets , and recombinant human HGF was purchased from R & D Systems (Minneapolis, Minn.).

    Techniques: Lysis, Activity Assay, Incubation, Staining

    Effects of TGF-β and HGF on cell cycle distribution of Mv1Lu cells. Exponentially growing Mv1Lu cells were treated with 100 pM TGF-β, 220 pM HGF, or both in DMEM containing 10% FCS for 16 h. (A) Flow cytometry analysis. Inset: percentages of cells in different cell cycle phases. 2 n and 4 n , diploid and tetraploid DNA contents, respectively. (B) 5-BrdUrd incorporation. Cells were labeled with 5-BrdUrd during the last hour of the 16-h incubation, and 5-BrdUrd incorporation was detected by immunostaining. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei. The results are averages of five independent experiments; standard deviations are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Hepatocyte Growth Factor Releases Mink Epithelial Cells from Transforming Growth Factor ?1-Induced Growth Arrest by Restoring Cdk6 Expression and Cyclin E-Associated Cdk2 Activity

    doi:

    Figure Lengend Snippet: Effects of TGF-β and HGF on cell cycle distribution of Mv1Lu cells. Exponentially growing Mv1Lu cells were treated with 100 pM TGF-β, 220 pM HGF, or both in DMEM containing 10% FCS for 16 h. (A) Flow cytometry analysis. Inset: percentages of cells in different cell cycle phases. 2 n and 4 n , diploid and tetraploid DNA contents, respectively. (B) 5-BrdUrd incorporation. Cells were labeled with 5-BrdUrd during the last hour of the 16-h incubation, and 5-BrdUrd incorporation was detected by immunostaining. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei. The results are averages of five independent experiments; standard deviations are shown.

    Article Snippet: In all assays, except when otherwise stated, the cells were treated with 100 pM TGF-β1, 220 pM HGF, or both in DMEM containing 10% FCS for 16 h. TGF-β1 was purified from outdated human platelets , and recombinant human HGF was purchased from R & D Systems (Minneapolis, Minn.).

    Techniques: Flow Cytometry, Cytometry, Labeling, Incubation, Immunostaining, Staining

    Effects of TGF-β and HGF on phosphorylation of pRb. Mv1Lu cells were treated with TGF-β (100 pM) and HGF (220 pM), as indicated, and incubated for 16 h, and the cells were lysed with Laemmli sample buffer. Total cellular lysates (10 μg) were separated by SDS–7.5% PAGE and transferred to an Immobilon-P membrane, followed by immunoblotting against pRb. The hypophosphorylated (pRb hypo-P ) and hyperphosphorylated (pRb P ) forms of pRb are indicated (arrows).

    Journal: Molecular and Cellular Biology

    Article Title: Hepatocyte Growth Factor Releases Mink Epithelial Cells from Transforming Growth Factor ?1-Induced Growth Arrest by Restoring Cdk6 Expression and Cyclin E-Associated Cdk2 Activity

    doi:

    Figure Lengend Snippet: Effects of TGF-β and HGF on phosphorylation of pRb. Mv1Lu cells were treated with TGF-β (100 pM) and HGF (220 pM), as indicated, and incubated for 16 h, and the cells were lysed with Laemmli sample buffer. Total cellular lysates (10 μg) were separated by SDS–7.5% PAGE and transferred to an Immobilon-P membrane, followed by immunoblotting against pRb. The hypophosphorylated (pRb hypo-P ) and hyperphosphorylated (pRb P ) forms of pRb are indicated (arrows).

    Article Snippet: In all assays, except when otherwise stated, the cells were treated with 100 pM TGF-β1, 220 pM HGF, or both in DMEM containing 10% FCS for 16 h. TGF-β1 was purified from outdated human platelets , and recombinant human HGF was purchased from R & D Systems (Minneapolis, Minn.).

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis

    (A) Growth factor regulation of cyclin E-associated kinase activity following p27 immunodepletion. Sparsely seeded cells were treated with TGF-β (100 pM) and HGF (220 pM), as indicated, followed by lysis in NP-40 buffer. Lysates were immunoprecipitated with cyclin E antibody (lanes 1 to 4) or with three subsequent cycles of polyclonal p27 antibody, followed by cyclin E immunoprecipitation (lanes 5 to 8). Kinase assays towards histone H1 were carried out as described in Materials and Methods. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum compared to control cells (set at 100) are indicated. (B) Effects of p27 immunodepletion steps on cyclin E, Cdk2, p27, and their complexes. In parallel with experiment represented by panel A, cell lysates before and after three immunodepletion cycles with anti-p27 antibody were analyzed by immunoprecipitation (IP) with the indicated antibody, followed by immunoblotting (WB). Total cyclin E and p27 were analyzed by immunoblotting. Fold changes compared to controls are shown below each analysis.

    Journal: Molecular and Cellular Biology

    Article Title: Hepatocyte Growth Factor Releases Mink Epithelial Cells from Transforming Growth Factor ?1-Induced Growth Arrest by Restoring Cdk6 Expression and Cyclin E-Associated Cdk2 Activity

    doi:

    Figure Lengend Snippet: (A) Growth factor regulation of cyclin E-associated kinase activity following p27 immunodepletion. Sparsely seeded cells were treated with TGF-β (100 pM) and HGF (220 pM), as indicated, followed by lysis in NP-40 buffer. Lysates were immunoprecipitated with cyclin E antibody (lanes 1 to 4) or with three subsequent cycles of polyclonal p27 antibody, followed by cyclin E immunoprecipitation (lanes 5 to 8). Kinase assays towards histone H1 were carried out as described in Materials and Methods. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum compared to control cells (set at 100) are indicated. (B) Effects of p27 immunodepletion steps on cyclin E, Cdk2, p27, and their complexes. In parallel with experiment represented by panel A, cell lysates before and after three immunodepletion cycles with anti-p27 antibody were analyzed by immunoprecipitation (IP) with the indicated antibody, followed by immunoblotting (WB). Total cyclin E and p27 were analyzed by immunoblotting. Fold changes compared to controls are shown below each analysis.

    Article Snippet: In all assays, except when otherwise stated, the cells were treated with 100 pM TGF-β1, 220 pM HGF, or both in DMEM containing 10% FCS for 16 h. TGF-β1 was purified from outdated human platelets , and recombinant human HGF was purchased from R & D Systems (Minneapolis, Minn.).

    Techniques: Activity Assay, Lysis, Immunoprecipitation, Western Blot

    Effects of TGF-β and HGF on expression and complex formation of Cdk2 and cyclin E. Mv1Lu cells were treated with the indicated growth factors, and cell lysates were prepared. (A) Immunoblotting analysis of Cdk2 and cyclin E. The different forms of Cdk2 are indicated (Thr 160 phosphorylated form of Cdk2 is designated cdk2 T160P ). (B) Complex formation of cyclin E and Cdk2 was detected by immunoprecipitation (IP), followed by Western blotting (WB) analysis with the indicated antibodies. Fold changes compared to controls are shown below each analysis.

    Journal: Molecular and Cellular Biology

    Article Title: Hepatocyte Growth Factor Releases Mink Epithelial Cells from Transforming Growth Factor ?1-Induced Growth Arrest by Restoring Cdk6 Expression and Cyclin E-Associated Cdk2 Activity

    doi:

    Figure Lengend Snippet: Effects of TGF-β and HGF on expression and complex formation of Cdk2 and cyclin E. Mv1Lu cells were treated with the indicated growth factors, and cell lysates were prepared. (A) Immunoblotting analysis of Cdk2 and cyclin E. The different forms of Cdk2 are indicated (Thr 160 phosphorylated form of Cdk2 is designated cdk2 T160P ). (B) Complex formation of cyclin E and Cdk2 was detected by immunoprecipitation (IP), followed by Western blotting (WB) analysis with the indicated antibodies. Fold changes compared to controls are shown below each analysis.

    Article Snippet: In all assays, except when otherwise stated, the cells were treated with 100 pM TGF-β1, 220 pM HGF, or both in DMEM containing 10% FCS for 16 h. TGF-β1 was purified from outdated human platelets , and recombinant human HGF was purchased from R & D Systems (Minneapolis, Minn.).

    Techniques: Expressing, Immunoprecipitation, Western Blot

    The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, Sp1, p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P

    Journal: British Journal of Pharmacology

    Article Title: Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma, et al. Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma

    doi: 10.1111/bph.15046

    Figure Lengend Snippet: The in vivo suppression of orthotopic hepatocellular carcinoma (HCC) growth and lung metastasis via geniposide is mediated by the TLR4/MyD88 pathway. (a) Representative figures of orthotopic growth of MHCC‐97L cells expressing luciferase reporter (uppermost panel) from mice with various treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 in a single injection), respectively. Line chart and histogram in the middle panel illustrate the luciferase signal intensities of HCC in either primary (left) or metastatic regions in the lung (right), respectively. The representative image showing lung metastasis of luciferase‐labelled MHCC‐97L cells is in the lower panel. Tumour growth was monitored by luciferase imaging in live animals, once a week. (b) Representative images of HCC in the liver from control or LPS‐injected mice with geniposide treatment. Both the weights of HCC with liver tissue and HCC volume were measured as shown in the right panel, respectively. (c) Representative graphs show haematoxylin and eosin staining of orthotopic HCC developed in the livers and lungs from mice with different interventions. A boundary between tumour and normal liver is clearly noticeable in the group treated with geniposide alone. Quantification of pulmonary nodules is presented in the bar chart (lower panel). (d) Pulmonary metastasis of HCC identified by glypican‐3/DAPI co‐localization. Glypican‐3‐positive cells in the lungs of each group are quantified. (e) Mitotic events in HCC are presented in the left panel. Quantification of the mitotic index is shown in the right panel. (f) HCC‐derived in vivo protein expression of TLR4, Sp1, p‐STAT3 and p65 in the HCC mouse model with diversified treatments. (g) Serum VEGF expression in orthotopic HCC mice with three independent treatments, including vehicle, geniposide (30 mg·kg −1 for 2 days) and geniposide with LPS (3 mg·kg −1 per single injection), respectively. (h) Representative immunofluorescence graphs of hepatic tumour tissues from HCC‐bearing mice with various treatments. Inset images in the right corner are the respectively enlarged fields with merged signals, showing that either CD31 (red) or MyD88 (green) is overlapped with DAPI staining (blue). Note that targeting HCC angiogenesis by geniposide (30 mg·kg −1 for 2 days) is associated with the direct shutdown of the TLR4/MyD88‐dependent pathway, which was reversed by the additional administration of LPS (3 mg·kg −1 in a single injection). These results demonstrated that geniposide can significantly repress HCC proliferation, angiogenesis and pulmonary metastasis by down‐regulating the TLR4/MyD88 signalling pathway. All data indicated are means ± SD of five independent experiments with at least three replicates. * P

    Article Snippet: After finishing the electro‐transfer, membranes were immersed in the blocking buffer containing 5% BSA for 2 h prior to the incubation with primary antibodies, including anti‐TLR4 (Cat: MAB6248, R & D Systems, USA), anti‐MD2 (Cat: ab24182, Abcam, USA), anti‐glypican‐3 (Cat: ab66596, Abcam, USA), anti‐Sp1 (Cat: 9389, CST, USA), anti‐STAT3 (Cat: 9139, CST, USA), anti‐phospho‐STAT3 (Cat: 9145, CST, USA), anti‐HIF‐1α (Cat: 36169, CST, USA), anti‐phospho‐p38 MAPK (Cat: 4511, CST, USA), anti‐IκB‐α (Cat: 4812, CST, USA), anti‐p65 (Cat: 8242, CST, USA) and anti‐MyD88 (Cat: 50010, CST, USA), respectively.

    Techniques: In Vivo, Expressing, Luciferase, Mouse Assay, Injection, Imaging, Staining, Derivative Assay, Immunofluorescence

    Direct inhibition of the TLR4/MyD88 pathway is involved in the anti‐angiogenic actions of geniposide in hepatocellular carcinoma (HCC). (a) 3D structure of binding profile between TLR4 and geniposide is visualized and analysed by in silico molecular docking approach. Magnified views are the potential binding clusters (left panel). The central figure is the sensorgram (red curves) for the binding affinity analysis of geniposide with various concentrations (0–12.8 μM) over a TLR4‐immobilized CM5 sensor chip, which is analysed by surface plasmon resonance (SPR) technology, while the right inset plot (black line) represents the response intensity of 0–12.8‐μM geniposide to TLR4. The linear regression is plotted by a four‐parameter logistic equation ( R 2 = 0.952, K D = 6.716e −6 M) by the BIAevaluation system (GE Healthcare, Sweden). (b) The interaction of MD2 and TLR4 was measured by co‐immunoprecipitation assay and immunoblotting (upper panel), while the lower panel is the mRNA expression of TLR4 in both MHCC‐97L and PLC/PRF/5 cells under geniposide treatment in normoxic condition. (c, d) Protein expressions of TLR4/MyD88 and its downstream factors p‐p38 MAPK, p65 and IκB‐α in both 24‐h geniposide‐treated MHCC‐97L and PLC/PRF/5 under either normoxic or hypoxic condition. (e) Protein levels of TLR4, MyD88, Sp1, STAT3, p‐STAT3, p‐p38 MAPK and p65 in MHCC‐97L and PLC/PRF/5 cells under co‐treatment of geniposide and LPS (100 ng·ml −1 ). (f, g) Secretion and mRNA activity of VEGF in both MHCC‐97L and PLC/PRF/5 cells upon the cooperative intervention of geniposide and LPS (100 ng·ml −1 ) under normoxic or hypoxic environment, analysed by elisa assay and RT‐PCR, respectively. (h) Determination of HCC‐derived tube formation of HUVECs via either single or co‐treatments as shown in the left panel, including geniposide (200 μg·ml −1 ), LPS (100 ng·ml −1 ) and recombinant VEGF (re‐VEGF; 20 ng·ml −1 ). Quantification of tubular networks is shown in the bar charts (lower panel). (i) The migration of HUVECs is significantly reduced when cultured in the supernatant derived from geniposide‐treated PLC/PRF/5 cells under normoxic conditions for 24 h. Note that the TLR4/MyD88 signalling pathway can be down‐regulated by geniposide treatment, which is caused by geniposide‐induced direct inhibition of TLR4 protein. Also, the addition of either LPS or recombinant VEGF leads to significant reversal of the anti‐angiogenic effect of geniposide. All data are presented as mean ± SD of five independent experiments with at least three replicates. * P

    Journal: British Journal of Pharmacology

    Article Title: Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma, et al. Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma

    doi: 10.1111/bph.15046

    Figure Lengend Snippet: Direct inhibition of the TLR4/MyD88 pathway is involved in the anti‐angiogenic actions of geniposide in hepatocellular carcinoma (HCC). (a) 3D structure of binding profile between TLR4 and geniposide is visualized and analysed by in silico molecular docking approach. Magnified views are the potential binding clusters (left panel). The central figure is the sensorgram (red curves) for the binding affinity analysis of geniposide with various concentrations (0–12.8 μM) over a TLR4‐immobilized CM5 sensor chip, which is analysed by surface plasmon resonance (SPR) technology, while the right inset plot (black line) represents the response intensity of 0–12.8‐μM geniposide to TLR4. The linear regression is plotted by a four‐parameter logistic equation ( R 2 = 0.952, K D = 6.716e −6 M) by the BIAevaluation system (GE Healthcare, Sweden). (b) The interaction of MD2 and TLR4 was measured by co‐immunoprecipitation assay and immunoblotting (upper panel), while the lower panel is the mRNA expression of TLR4 in both MHCC‐97L and PLC/PRF/5 cells under geniposide treatment in normoxic condition. (c, d) Protein expressions of TLR4/MyD88 and its downstream factors p‐p38 MAPK, p65 and IκB‐α in both 24‐h geniposide‐treated MHCC‐97L and PLC/PRF/5 under either normoxic or hypoxic condition. (e) Protein levels of TLR4, MyD88, Sp1, STAT3, p‐STAT3, p‐p38 MAPK and p65 in MHCC‐97L and PLC/PRF/5 cells under co‐treatment of geniposide and LPS (100 ng·ml −1 ). (f, g) Secretion and mRNA activity of VEGF in both MHCC‐97L and PLC/PRF/5 cells upon the cooperative intervention of geniposide and LPS (100 ng·ml −1 ) under normoxic or hypoxic environment, analysed by elisa assay and RT‐PCR, respectively. (h) Determination of HCC‐derived tube formation of HUVECs via either single or co‐treatments as shown in the left panel, including geniposide (200 μg·ml −1 ), LPS (100 ng·ml −1 ) and recombinant VEGF (re‐VEGF; 20 ng·ml −1 ). Quantification of tubular networks is shown in the bar charts (lower panel). (i) The migration of HUVECs is significantly reduced when cultured in the supernatant derived from geniposide‐treated PLC/PRF/5 cells under normoxic conditions for 24 h. Note that the TLR4/MyD88 signalling pathway can be down‐regulated by geniposide treatment, which is caused by geniposide‐induced direct inhibition of TLR4 protein. Also, the addition of either LPS or recombinant VEGF leads to significant reversal of the anti‐angiogenic effect of geniposide. All data are presented as mean ± SD of five independent experiments with at least three replicates. * P

    Article Snippet: After finishing the electro‐transfer, membranes were immersed in the blocking buffer containing 5% BSA for 2 h prior to the incubation with primary antibodies, including anti‐TLR4 (Cat: MAB6248, R & D Systems, USA), anti‐MD2 (Cat: ab24182, Abcam, USA), anti‐glypican‐3 (Cat: ab66596, Abcam, USA), anti‐Sp1 (Cat: 9389, CST, USA), anti‐STAT3 (Cat: 9139, CST, USA), anti‐phospho‐STAT3 (Cat: 9145, CST, USA), anti‐HIF‐1α (Cat: 36169, CST, USA), anti‐phospho‐p38 MAPK (Cat: 4511, CST, USA), anti‐IκB‐α (Cat: 4812, CST, USA), anti‐p65 (Cat: 8242, CST, USA) and anti‐MyD88 (Cat: 50010, CST, USA), respectively.

    Techniques: Inhibition, Binding Assay, In Silico, Chromatin Immunoprecipitation, SPR Assay, Co-Immunoprecipitation Assay, Expressing, Planar Chromatography, Activity Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Recombinant, Migration, Cell Culture

    Geniposide down‐regulated Sp1‐ and STAT3‐related VEGF transcription in hepatocellular carcinoma (HCC) cells. (a) Binding sites of putative transcription factors (Sp1 and STAT3) in the specific sequence of luciferase‐targeted VEGF promotor. (b) Determination of expressions of Sp1, total and phosphorylated STAT3 in MHCC‐97L and PLC/PRF/5 cells with or without geniposide stimulation under normoxic or hypoxic condition. (c) Representative confocal images of in vitro HCC cells with or without geniposide. HCC slices are incubated with both antibodies, Sp1 and STAT3. Merged figures illustrate the co‐localization of Sp1 (green), STAT3 (red) and cell nucleus (blue). (d) Normoxic mRNA level or (e) secretory expression of VEGF in MHCC‐97L and PLC/PRF/5 cells with three independent conditions by plasmid transfection as follows: overexpression of Sp1, STAT3 and dual hyperactivation of Sp1 and STAT3, respectively. Note that the expression of Sp1 and STAT3 can be markedly inhibited by geniposide intervention. All data are shown as mean ± SD of five independent assays with at least three replicates. * P

    Journal: British Journal of Pharmacology

    Article Title: Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma, et al. Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma

    doi: 10.1111/bph.15046

    Figure Lengend Snippet: Geniposide down‐regulated Sp1‐ and STAT3‐related VEGF transcription in hepatocellular carcinoma (HCC) cells. (a) Binding sites of putative transcription factors (Sp1 and STAT3) in the specific sequence of luciferase‐targeted VEGF promotor. (b) Determination of expressions of Sp1, total and phosphorylated STAT3 in MHCC‐97L and PLC/PRF/5 cells with or without geniposide stimulation under normoxic or hypoxic condition. (c) Representative confocal images of in vitro HCC cells with or without geniposide. HCC slices are incubated with both antibodies, Sp1 and STAT3. Merged figures illustrate the co‐localization of Sp1 (green), STAT3 (red) and cell nucleus (blue). (d) Normoxic mRNA level or (e) secretory expression of VEGF in MHCC‐97L and PLC/PRF/5 cells with three independent conditions by plasmid transfection as follows: overexpression of Sp1, STAT3 and dual hyperactivation of Sp1 and STAT3, respectively. Note that the expression of Sp1 and STAT3 can be markedly inhibited by geniposide intervention. All data are shown as mean ± SD of five independent assays with at least three replicates. * P

    Article Snippet: After finishing the electro‐transfer, membranes were immersed in the blocking buffer containing 5% BSA for 2 h prior to the incubation with primary antibodies, including anti‐TLR4 (Cat: MAB6248, R & D Systems, USA), anti‐MD2 (Cat: ab24182, Abcam, USA), anti‐glypican‐3 (Cat: ab66596, Abcam, USA), anti‐Sp1 (Cat: 9389, CST, USA), anti‐STAT3 (Cat: 9139, CST, USA), anti‐phospho‐STAT3 (Cat: 9145, CST, USA), anti‐HIF‐1α (Cat: 36169, CST, USA), anti‐phospho‐p38 MAPK (Cat: 4511, CST, USA), anti‐IκB‐α (Cat: 4812, CST, USA), anti‐p65 (Cat: 8242, CST, USA) and anti‐MyD88 (Cat: 50010, CST, USA), respectively.

    Techniques: Binding Assay, Sequencing, Luciferase, Planar Chromatography, In Vitro, Incubation, Expressing, Plasmid Preparation, Transfection, Over Expression

    Proposed mechanisms underlying the positive therapeutic effects of combined melatonin and exendin-4 therapy on reversing the deterioration of kidney function from cardiorenal syndrome (CRS). LEVF = left ventricular ejection fraction; TNF-α = tumor necrosis factor alpha; NF-κB = nuclear factor-κB; MMP-9 = matrix metalloproteinase 9; iNOS = inducible nitric oxide synthase; HO-1 = heme oxygenase 1; TGF-β = transforming growth factor beta; c-PARP = cleaved Poly (ADP-ribose) polymerase.

    Journal: American Journal of Translational Research

    Article Title: Combined therapy with melatonin and exendin-4 effectively attenuated the deterioration of renal function in rat cardiorenal syndrome

    doi:

    Figure Lengend Snippet: Proposed mechanisms underlying the positive therapeutic effects of combined melatonin and exendin-4 therapy on reversing the deterioration of kidney function from cardiorenal syndrome (CRS). LEVF = left ventricular ejection fraction; TNF-α = tumor necrosis factor alpha; NF-κB = nuclear factor-κB; MMP-9 = matrix metalloproteinase 9; iNOS = inducible nitric oxide synthase; HO-1 = heme oxygenase 1; TGF-β = transforming growth factor beta; c-PARP = cleaved Poly (ADP-ribose) polymerase.

    Article Snippet: The membranes were incubated with the indicated primary antibodies [NOX-1 (1:1500, Sigma, St. Louis, MO), NOX-2 (1:1500, Sigma, MO), NOX-4 (1:1000, Abcam, Cambridge, MA), cleaved caspase 3 (1:1000, Cell Signaling, Danvers, MA), cleaved Poly (ADP-ribose) polymerase (PARP) (1:1000, Cell Signaling, Danvers, MA), γ-H2AX (1:1000, Cell Signaling, Danvers, MA), glucagon-like-peptide-1 receptor (GLP-1R) (1:1000, Abcam, Cambridge, MA), interleukin (IL)-1β (1:1000, Cell Signaling, Danvers, MA), tumor necrosis factor (TNF)-α (1:1000, Cell Signaling, Danvers, MA), nuclear factor (NF)-κB (1:600, Abcam, Cambridge, MA), MMP-9 (1:3000, Abcam, Cambridge, MA), Bax (1:1000, Abcam, Cambridge, MA), Smad3 (1:1000, Cell Signaling, Danvers, MA), Smad1/5 (1:1000, Cell Signaling, Danvers, MA), transforming growth factor (TGF)-β (1:1000, Abcam, Cambridge, MA), RANTES (1:1000, Cell Signaling, Danvers, MA), inducible nitric oxide synthase (iNOS) (1:250, Abcam, Cambridge, MA) and actin (1:10000, Chemicon, Billerica, MA)] for 1 hour at room temperature.

    Techniques:

    FoxO1-S253 A/A mice exhibit impaired glucagon tolerance test and hepatic gene expression. (A) Glycogen concentration in the liver of mice at 16-hour fasting and random-fed states. * P

    Journal: Endocrinology

    Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

    doi: 10.1210/en.2018-00853

    Figure Lengend Snippet: FoxO1-S253 A/A mice exhibit impaired glucagon tolerance test and hepatic gene expression. (A) Glycogen concentration in the liver of mice at 16-hour fasting and random-fed states. * P

    Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology , Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( )], β-actin [catalog no. 4970; Cell Signaling Technology ( )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology , Dallas, TX] were used for Western blot.

    Techniques: Mouse Assay, Expressing, Concentration Assay

    EE increases in FoxO1-S253 A/A mice. The mice at the age of 16 weeks old were placed in metabolic cages for the measurement of food intake, physical activity, and VO 2 , and EE. (A) Food intake, (B) physical activity, (C) VO 2 , (D) EE, and (E) RER were calculated. # P

    Journal: Endocrinology

    Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

    doi: 10.1210/en.2018-00853

    Figure Lengend Snippet: EE increases in FoxO1-S253 A/A mice. The mice at the age of 16 weeks old were placed in metabolic cages for the measurement of food intake, physical activity, and VO 2 , and EE. (A) Food intake, (B) physical activity, (C) VO 2 , (D) EE, and (E) RER were calculated. # P

    Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology , Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( )], β-actin [catalog no. 4970; Cell Signaling Technology ( )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology , Dallas, TX] were used for Western blot.

    Techniques: Mouse Assay, Activity Assay

    FoxO1-S253 A/A mice exhibit impaired α -cells, insulin, and glucagon synthesis in the pancreas. (A) Representative pancreas of control (WT) and A/A mice at the age of 12 weeks at random-fed states. * P

    Journal: Endocrinology

    Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

    doi: 10.1210/en.2018-00853

    Figure Lengend Snippet: FoxO1-S253 A/A mice exhibit impaired α -cells, insulin, and glucagon synthesis in the pancreas. (A) Representative pancreas of control (WT) and A/A mice at the age of 12 weeks at random-fed states. * P

    Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology , Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( )], β-actin [catalog no. 4970; Cell Signaling Technology ( )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology , Dallas, TX] were used for Western blot.

    Techniques: Mouse Assay

    Generation of FoxO1-S253 A/A mice and postprandial hyperglycemia. (A) The genomic locus of FoxO1 in mouse genome. (B) Gene-targeting strategy for generation of FoxO1-S253A mutation via embryonic stem (ES) cell and homologous recombination (HR). (C and D) Southern blot was performed for screening the 5′- and 3′-end HR in FoxO1 genomic loci. DNA sequencing of endogenous FoxO1 loci of negative and positive ES cells. (E) Body weight curve of mice of control wild-type (WT), heterozygous (A/+), and homozygous (A/A) mice at the ages of 4 to 16 weeks. (F) Effect of insulin on FoxO1 protein and phosphorylation (p) in hepatocytes. Primary hepatocytes were isolated from the control and A/A mice and treated with 100 nM insulin for 30 minutes, and 100 µg protein of cell lysates was subjected to Western blot against the antibody of FoxO1, T24, S316, and β -actin. (G and H) Blood glucose levels were measured in WT, A/+, and A/A mice at the ages 4 to 16 weeks during (G) 16 hours fasting and (H) random-fed conditions. * P

    Journal: Endocrinology

    Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

    doi: 10.1210/en.2018-00853

    Figure Lengend Snippet: Generation of FoxO1-S253 A/A mice and postprandial hyperglycemia. (A) The genomic locus of FoxO1 in mouse genome. (B) Gene-targeting strategy for generation of FoxO1-S253A mutation via embryonic stem (ES) cell and homologous recombination (HR). (C and D) Southern blot was performed for screening the 5′- and 3′-end HR in FoxO1 genomic loci. DNA sequencing of endogenous FoxO1 loci of negative and positive ES cells. (E) Body weight curve of mice of control wild-type (WT), heterozygous (A/+), and homozygous (A/A) mice at the ages of 4 to 16 weeks. (F) Effect of insulin on FoxO1 protein and phosphorylation (p) in hepatocytes. Primary hepatocytes were isolated from the control and A/A mice and treated with 100 nM insulin for 30 minutes, and 100 µg protein of cell lysates was subjected to Western blot against the antibody of FoxO1, T24, S316, and β -actin. (G and H) Blood glucose levels were measured in WT, A/+, and A/A mice at the ages 4 to 16 weeks during (G) 16 hours fasting and (H) random-fed conditions. * P

    Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology , Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( )], β-actin [catalog no. 4970; Cell Signaling Technology ( )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology , Dallas, TX] were used for Western blot.

    Techniques: Mouse Assay, Mutagenesis, Homologous Recombination, Southern Blot, DNA Sequencing, Isolation, Western Blot

    Dephosphorylation of FoxO1-S253 in FoxO1-S253 A/A mice regulates hepatic gluconeogenesis, glycogen metabolism, and pancreatic plasticity. (A) FoxO1-S253 de-phosphorylation controls insulin and glucagon production in the pancreas, regulating liver glucose metabolism in the feeding state, resulting in an increase of blood glucose. (B) At the fasting state, where blood glucagon decreases, reducing the blood glucose. →, stimulation; ˧, inhibition.

    Journal: Endocrinology

    Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

    doi: 10.1210/en.2018-00853

    Figure Lengend Snippet: Dephosphorylation of FoxO1-S253 in FoxO1-S253 A/A mice regulates hepatic gluconeogenesis, glycogen metabolism, and pancreatic plasticity. (A) FoxO1-S253 de-phosphorylation controls insulin and glucagon production in the pancreas, regulating liver glucose metabolism in the feeding state, resulting in an increase of blood glucose. (B) At the fasting state, where blood glucagon decreases, reducing the blood glucose. →, stimulation; ˧, inhibition.

    Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology , Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( )], β-actin [catalog no. 4970; Cell Signaling Technology ( )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology , Dallas, TX] were used for Western blot.

    Techniques: De-Phosphorylation Assay, Mouse Assay, Inhibition

    Action of conditioned medium on Akt, ERK, AS160, and Akt substrates phosphorylation after glucose stimulation, or IRS-1 and -2 protein expression in rat primary β-cells. Conditioned media and abbreviations as described in the legend to Fig. 3 . After 24-h culture in conditioned medium, β-cells were incubated 1 h at 2.8 mmol/L or 16.7 mmol/L glucose (Glc). Open bars = 2.8 mmol/L glucose; closed bars = 16.7 mmol/L glucose. Western blots were scanned and data normalized to total protein or actin as indicated. N = 3 independent experiments. A : Akt Ser 473 phosphorylation. * P

    Journal: Diabetes

    Article Title: Bimodal Effect on Pancreatic ?-Cells of Secretory Products From Normal or Insulin-Resistant Human Skeletal Muscle

    doi: 10.2337/db10-1178

    Figure Lengend Snippet: Action of conditioned medium on Akt, ERK, AS160, and Akt substrates phosphorylation after glucose stimulation, or IRS-1 and -2 protein expression in rat primary β-cells. Conditioned media and abbreviations as described in the legend to Fig. 3 . After 24-h culture in conditioned medium, β-cells were incubated 1 h at 2.8 mmol/L or 16.7 mmol/L glucose (Glc). Open bars = 2.8 mmol/L glucose; closed bars = 16.7 mmol/L glucose. Western blots were scanned and data normalized to total protein or actin as indicated. N = 3 independent experiments. A : Akt Ser 473 phosphorylation. * P

    Article Snippet: The antibodies and reagents were as follows: human TNF (hTNF)-α (R & D Systems Europe Ltd., Abingdon, U.K.); anti-phospho–Akt-(Ser473), anti-phospho–ERK-1/2-(Thr202/Tyr204) (New England Biolabs, Beverly, MA); anti-phospho–(Ser/Thr)-AS160, anti-AS160, anti-Akt, anti-ERK, anti–insulin receptor substrate (IRS)-2 (Cell Signaling Technology, Beverly, MA); antiactin (Sigma, Buchs, Switzerland); anti–IRS-1 (gift from Dr. M.F.

    Techniques: Expressing, Incubation, Gas Chromatography, Western Blot

    Establishment of a murine breast cancer cell line undergoing TGFβ-induced EMT. ( A ) Primary tumor cells were isolated from an advanced breast tumor of a MMTV-PyMT transgenic female mouse and were cultured for at least 2 months prior to further experimentation, resulting in a novel cell line termed Py2T. ( B ) Py2T cells maintain the MMTV-PyMT transgene. The MMTV-PyMT transgene was detected by PCR and agarose gel electrophoresis. DNA from an MMTV-PyMT tumor and from normal murine mammary gland (NMuMG) cells served as positive and negative controls, respectively. ( C ) Py2T cells lost the expression of the MMTV-PyMT transgene. Immunoblotting for the PyMT protein was performed on lysates of Py2T cells untreated or treated with 0.1 µM Dexamethasone for up to 72 h to induce the MMTV promoter. Lysates of an MMTV-PyMT tumor and NMuMG cells served as positive and negative controls, respectively. ( D ) Treatment of Py2T cells with known EMT inducers. Cells were continuously treated with the indicated growth factors and cytokines for 10 days (2 ng/mL TGFβ1; 50 ng/mL EGF; 10 ng/mL IGF-I; 50 ng/mL HGF; 20 ng/mL FGF-2; 20 ng/mL PDGF-BB; 50 ng/mL IL-6). Potential morphological changes were analyzed by phase-contrast microscopy. ( E ) Expression of epithelial (E-cadherin) and mesenchymal (N-cadherin, fibronectin) markers were analyzed by immunoblotting of the lysates of cells treated in (D). ( F ) Immunoblotting analysis of EMT marker expression in Py2T and Py2T LT cells. The mesenchymal subline Py2T LT (long-term) was generated by TGFβ-treatment of Py2T cells for at least 20 days, and was subsequently maintained in TGFβ containing growth medium. ( G ) Analysis of markers for EMT and breast cell type before and after TGFβ-induced EMT. Immunofluorescence staining was performed with antibodies against E-Cadherin (epithelial marker), vimentin (mesenchymal marker), estrogen receptor alpha (ERα), cytokeratin 8/18 (luminal markers) and cytokeratin 14 (basal marker). Scale bar, 20 µm.

    Journal: PLoS ONE

    Article Title: Py2T Murine Breast Cancer Cells, a Versatile Model of TGF?-Induced EMT In Vitro and In Vivo

    doi: 10.1371/journal.pone.0048651

    Figure Lengend Snippet: Establishment of a murine breast cancer cell line undergoing TGFβ-induced EMT. ( A ) Primary tumor cells were isolated from an advanced breast tumor of a MMTV-PyMT transgenic female mouse and were cultured for at least 2 months prior to further experimentation, resulting in a novel cell line termed Py2T. ( B ) Py2T cells maintain the MMTV-PyMT transgene. The MMTV-PyMT transgene was detected by PCR and agarose gel electrophoresis. DNA from an MMTV-PyMT tumor and from normal murine mammary gland (NMuMG) cells served as positive and negative controls, respectively. ( C ) Py2T cells lost the expression of the MMTV-PyMT transgene. Immunoblotting for the PyMT protein was performed on lysates of Py2T cells untreated or treated with 0.1 µM Dexamethasone for up to 72 h to induce the MMTV promoter. Lysates of an MMTV-PyMT tumor and NMuMG cells served as positive and negative controls, respectively. ( D ) Treatment of Py2T cells with known EMT inducers. Cells were continuously treated with the indicated growth factors and cytokines for 10 days (2 ng/mL TGFβ1; 50 ng/mL EGF; 10 ng/mL IGF-I; 50 ng/mL HGF; 20 ng/mL FGF-2; 20 ng/mL PDGF-BB; 50 ng/mL IL-6). Potential morphological changes were analyzed by phase-contrast microscopy. ( E ) Expression of epithelial (E-cadherin) and mesenchymal (N-cadherin, fibronectin) markers were analyzed by immunoblotting of the lysates of cells treated in (D). ( F ) Immunoblotting analysis of EMT marker expression in Py2T and Py2T LT cells. The mesenchymal subline Py2T LT (long-term) was generated by TGFβ-treatment of Py2T cells for at least 20 days, and was subsequently maintained in TGFβ containing growth medium. ( G ) Analysis of markers for EMT and breast cell type before and after TGFβ-induced EMT. Immunofluorescence staining was performed with antibodies against E-Cadherin (epithelial marker), vimentin (mesenchymal marker), estrogen receptor alpha (ERα), cytokeratin 8/18 (luminal markers) and cytokeratin 14 (basal marker). Scale bar, 20 µm.

    Article Snippet: Antibodies and Reagents Antibodies: PyMT (mouse monoclonal Pab762, a kind gift of Dr. S. Dilworth, Imperial College London), Actin (sc-1616, SantaCruz Biotechnology), E-cadherin (610182, Transduction Laboratories, used for immunoblotting and IHC), E-cadherin (13-1900, Zymed, used for immunofluorescence stainings), N-cadherin (M142, Takara Bio), fibronectin (F3648 Sigma-Aldrich), GAPDH (ab9485, Abcam), cytokeratin 14 (RB-9020-P0, NeoMarkers), cytokeratin 8/18 (20R-CP004, Fitzgerald), vimentin (V2258, Sigma-Aldrich), ERα (sc-542, Santa Cruz Biotechnology), ZO-1 (617300, Zymed), F4/80 (MCAP497, Serotec), CD45 (550539, BD), Smad2/3 (610842, BD), Smad3 pSer423/425 (9520, Cell Signalling), β-catenin (C2206, Sigma-Aldrich), Smad4 (sc-7966, Santa Cruz Biotechnology), RhoA (sc-418, Santa Cruz Biotechnology).

    Techniques: Isolation, Transgenic Assay, Cell Culture, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Microscopy, Marker, Generated, Immunofluorescence, Staining

    Orthotopic transplantation of Py2T cells into syngeneic mice results in the formation of invasive tumors. ( A ) H E staining of histological sections from tumors of MMTV-PyMT transgenic mice and from transplanted Py2T tumors. 1×10 6 Py2T cells were transplanted into the fat pad of 8 weeks old female FVB/N mice and allowed to grow tumors for 27 days. Late-stage MMTV-PyMT tumors were from 12 weeks old female mice. Bottom panels : enlarged regions indicated by the white squares in the top panels. Note the typical pushing borders in MMTV-PyMT tumors in contrast to stream-like invasion of fat tissue in Py2T tumors. Scale bars, 200 µm. ( B ) Polyoma-middle-T (PyMT) expression in MMTV-PyMT and Py2T tumors. Paraffin sections were stained with an antibody against PyMT. Immunohistochemical staining in the absence of primary antibody (1°) was used as negative control. Scale bar, 100 µm. ( C ) Immunoblotting analysis for EMT markers in tumor lysates of MMTV-PyMT and Py2T tumors. Lysate from cultured Py2T cells is included as a control. Note the loss of E-cadherin expression and upregulation of mesenchymal markers (N-cadherin, fibronectin) in Py2T tumors.

    Journal: PLoS ONE

    Article Title: Py2T Murine Breast Cancer Cells, a Versatile Model of TGF?-Induced EMT In Vitro and In Vivo

    doi: 10.1371/journal.pone.0048651

    Figure Lengend Snippet: Orthotopic transplantation of Py2T cells into syngeneic mice results in the formation of invasive tumors. ( A ) H E staining of histological sections from tumors of MMTV-PyMT transgenic mice and from transplanted Py2T tumors. 1×10 6 Py2T cells were transplanted into the fat pad of 8 weeks old female FVB/N mice and allowed to grow tumors for 27 days. Late-stage MMTV-PyMT tumors were from 12 weeks old female mice. Bottom panels : enlarged regions indicated by the white squares in the top panels. Note the typical pushing borders in MMTV-PyMT tumors in contrast to stream-like invasion of fat tissue in Py2T tumors. Scale bars, 200 µm. ( B ) Polyoma-middle-T (PyMT) expression in MMTV-PyMT and Py2T tumors. Paraffin sections were stained with an antibody against PyMT. Immunohistochemical staining in the absence of primary antibody (1°) was used as negative control. Scale bar, 100 µm. ( C ) Immunoblotting analysis for EMT markers in tumor lysates of MMTV-PyMT and Py2T tumors. Lysate from cultured Py2T cells is included as a control. Note the loss of E-cadherin expression and upregulation of mesenchymal markers (N-cadherin, fibronectin) in Py2T tumors.

    Article Snippet: Antibodies and Reagents Antibodies: PyMT (mouse monoclonal Pab762, a kind gift of Dr. S. Dilworth, Imperial College London), Actin (sc-1616, SantaCruz Biotechnology), E-cadherin (610182, Transduction Laboratories, used for immunoblotting and IHC), E-cadherin (13-1900, Zymed, used for immunofluorescence stainings), N-cadherin (M142, Takara Bio), fibronectin (F3648 Sigma-Aldrich), GAPDH (ab9485, Abcam), cytokeratin 14 (RB-9020-P0, NeoMarkers), cytokeratin 8/18 (20R-CP004, Fitzgerald), vimentin (V2258, Sigma-Aldrich), ERα (sc-542, Santa Cruz Biotechnology), ZO-1 (617300, Zymed), F4/80 (MCAP497, Serotec), CD45 (550539, BD), Smad2/3 (610842, BD), Smad3 pSer423/425 (9520, Cell Signalling), β-catenin (C2206, Sigma-Aldrich), Smad4 (sc-7966, Santa Cruz Biotechnology), RhoA (sc-418, Santa Cruz Biotechnology).

    Techniques: Transplantation Assay, Mouse Assay, Staining, Transgenic Assay, Expressing, Immunohistochemistry, Negative Control, Cell Culture

    Changes of migratory and invasive properties of Py2T cells before, during and after TGFβ-induced EMT. ( A ) Boyden chamber migration and invasion assay. Cells were treated with TGFβ for the indicated times (LT = long term treatment, as described in Fig. 1F). 25'000 cells were seeded into migration or invasion chambers in duplicate in the absence or presence of TGFβ and allowed to pass through the membrane pores for 24 hours along an FBS gradient. Invasion chambers were pre-coated with growth-factor reduced Matrigel (BD BioCoat chambers). Cells that passed through the membrane pores were stained with crystal violet and photographed ( bottom panels ) and then counted ( top graphs ). Results are expressed as mean ± S.E.M of three independent experiments. ( B ) Scratch wound healing assay. Cells pre-treated with TGFβ or not as indicated were starved over night and scratch wounds were introduced into confluent monolayers. Scratch wound closure was monitored by an IncuCyte™ live cell imaging system. Black masking represents initial gap width at 0 hours. Note the collective, sheet-like wound closure by untreated Py2T cells in contrast to single cell wound infiltration of TGFβ-treated cells (also see Movies S1 and S2 for live imaging data of this experiment). ( C ) Morphology of epithelial Py2T cells and mesenchymal Py2T LT cells grown on plastic tissue culture dishes (2D) and in Matrigel (4 mg/ml; 3D). Structures were grown for 6 days, and stained directly in Matrigel with antibodies against epithelial E-cadherin and ZO-1 or against mesenchymal vimentin and fibronectin. Immunofluorescence images were acquired by confocal microscopy. Scale bars, 25 µm. ( D ) Three-dimensional reconstruction of confocal imaging stacks from cells grown in Matrigel as described in (A) (See also Movies S5 and S6 for rotating 3D models). Scale bars, 25 µm.

    Journal: PLoS ONE

    Article Title: Py2T Murine Breast Cancer Cells, a Versatile Model of TGF?-Induced EMT In Vitro and In Vivo

    doi: 10.1371/journal.pone.0048651

    Figure Lengend Snippet: Changes of migratory and invasive properties of Py2T cells before, during and after TGFβ-induced EMT. ( A ) Boyden chamber migration and invasion assay. Cells were treated with TGFβ for the indicated times (LT = long term treatment, as described in Fig. 1F). 25'000 cells were seeded into migration or invasion chambers in duplicate in the absence or presence of TGFβ and allowed to pass through the membrane pores for 24 hours along an FBS gradient. Invasion chambers were pre-coated with growth-factor reduced Matrigel (BD BioCoat chambers). Cells that passed through the membrane pores were stained with crystal violet and photographed ( bottom panels ) and then counted ( top graphs ). Results are expressed as mean ± S.E.M of three independent experiments. ( B ) Scratch wound healing assay. Cells pre-treated with TGFβ or not as indicated were starved over night and scratch wounds were introduced into confluent monolayers. Scratch wound closure was monitored by an IncuCyte™ live cell imaging system. Black masking represents initial gap width at 0 hours. Note the collective, sheet-like wound closure by untreated Py2T cells in contrast to single cell wound infiltration of TGFβ-treated cells (also see Movies S1 and S2 for live imaging data of this experiment). ( C ) Morphology of epithelial Py2T cells and mesenchymal Py2T LT cells grown on plastic tissue culture dishes (2D) and in Matrigel (4 mg/ml; 3D). Structures were grown for 6 days, and stained directly in Matrigel with antibodies against epithelial E-cadherin and ZO-1 or against mesenchymal vimentin and fibronectin. Immunofluorescence images were acquired by confocal microscopy. Scale bars, 25 µm. ( D ) Three-dimensional reconstruction of confocal imaging stacks from cells grown in Matrigel as described in (A) (See also Movies S5 and S6 for rotating 3D models). Scale bars, 25 µm.

    Article Snippet: Antibodies and Reagents Antibodies: PyMT (mouse monoclonal Pab762, a kind gift of Dr. S. Dilworth, Imperial College London), Actin (sc-1616, SantaCruz Biotechnology), E-cadherin (610182, Transduction Laboratories, used for immunoblotting and IHC), E-cadherin (13-1900, Zymed, used for immunofluorescence stainings), N-cadherin (M142, Takara Bio), fibronectin (F3648 Sigma-Aldrich), GAPDH (ab9485, Abcam), cytokeratin 14 (RB-9020-P0, NeoMarkers), cytokeratin 8/18 (20R-CP004, Fitzgerald), vimentin (V2258, Sigma-Aldrich), ERα (sc-542, Santa Cruz Biotechnology), ZO-1 (617300, Zymed), F4/80 (MCAP497, Serotec), CD45 (550539, BD), Smad2/3 (610842, BD), Smad3 pSer423/425 (9520, Cell Signalling), β-catenin (C2206, Sigma-Aldrich), Smad4 (sc-7966, Santa Cruz Biotechnology), RhoA (sc-418, Santa Cruz Biotechnology).

    Techniques: Migration, Invasion Assay, Staining, Wound Healing Assay, Live Cell Imaging, Imaging, Immunofluorescence, Confocal Microscopy

    Kinetics and reversibility of TGFβ-induced EMT in Py2T cells. ( A ) Morphological changes of Py2T cells during a time-course of TGFβ-treatment. Cells were cultured in growth medium containing TGFβ (2 ng/ml) and phase-contrast microscopy pictures were taken at the indicated times. ( B ) Immunoblotting analysis of lysates prepared from Py2T cells treated as in (A). The expression of epithelial (E-cadherin), mesenchymal (N-cadherin, fibronectin), luminal (CK8/18) and basal (CK14) markers was analyzed. ( C ) Changes in the expression of EMT markers during TGFβ-induced EMT of Py2T cells. Py2T cells were treated for 10 days with TGFβ as described in (A). RNA was extracted at the indicated time points of TGFβ-treatment and quantitative RT-PCR was performed with primers specific for the EMT markers indicated. Expression levels are shown as mean fold difference of untreated cells (0d) ± S.E.M of 5 independent experiments. ( D–E ) Reversibility of TGFβ-induced EMT. Py2T cells were treated with TGFβ for 30 days to induce EMT and were then further cultured without TGFβ for additional 30 days. Phase-contrast microscopy images were taken at the indicated time points (E). E-cadherin expression levels were analyzed throughout the experiment by immunoblotting (F).

    Journal: PLoS ONE

    Article Title: Py2T Murine Breast Cancer Cells, a Versatile Model of TGF?-Induced EMT In Vitro and In Vivo

    doi: 10.1371/journal.pone.0048651

    Figure Lengend Snippet: Kinetics and reversibility of TGFβ-induced EMT in Py2T cells. ( A ) Morphological changes of Py2T cells during a time-course of TGFβ-treatment. Cells were cultured in growth medium containing TGFβ (2 ng/ml) and phase-contrast microscopy pictures were taken at the indicated times. ( B ) Immunoblotting analysis of lysates prepared from Py2T cells treated as in (A). The expression of epithelial (E-cadherin), mesenchymal (N-cadherin, fibronectin), luminal (CK8/18) and basal (CK14) markers was analyzed. ( C ) Changes in the expression of EMT markers during TGFβ-induced EMT of Py2T cells. Py2T cells were treated for 10 days with TGFβ as described in (A). RNA was extracted at the indicated time points of TGFβ-treatment and quantitative RT-PCR was performed with primers specific for the EMT markers indicated. Expression levels are shown as mean fold difference of untreated cells (0d) ± S.E.M of 5 independent experiments. ( D–E ) Reversibility of TGFβ-induced EMT. Py2T cells were treated with TGFβ for 30 days to induce EMT and were then further cultured without TGFβ for additional 30 days. Phase-contrast microscopy images were taken at the indicated time points (E). E-cadherin expression levels were analyzed throughout the experiment by immunoblotting (F).

    Article Snippet: Antibodies and Reagents Antibodies: PyMT (mouse monoclonal Pab762, a kind gift of Dr. S. Dilworth, Imperial College London), Actin (sc-1616, SantaCruz Biotechnology), E-cadherin (610182, Transduction Laboratories, used for immunoblotting and IHC), E-cadherin (13-1900, Zymed, used for immunofluorescence stainings), N-cadherin (M142, Takara Bio), fibronectin (F3648 Sigma-Aldrich), GAPDH (ab9485, Abcam), cytokeratin 14 (RB-9020-P0, NeoMarkers), cytokeratin 8/18 (20R-CP004, Fitzgerald), vimentin (V2258, Sigma-Aldrich), ERα (sc-542, Santa Cruz Biotechnology), ZO-1 (617300, Zymed), F4/80 (MCAP497, Serotec), CD45 (550539, BD), Smad2/3 (610842, BD), Smad3 pSer423/425 (9520, Cell Signalling), β-catenin (C2206, Sigma-Aldrich), Smad4 (sc-7966, Santa Cruz Biotechnology), RhoA (sc-418, Santa Cruz Biotechnology).

    Techniques: Cell Culture, Microscopy, Expressing, Quantitative RT-PCR

    Identification of HMGB1 as a novel metformin-binding protein. A , structure of metformin ( upper ) and the biotinylated compound containing metformin-like biguanide structure ( lower ) used for the affinity chromatography. B , affinity purification was performed from rat liver cytosol with the metformin beads and the control biotin beads. Bead eluates were analyzed by SDS-PAGE followed by silver staining. Mass spectrometry analysis revealed the ∼25-kDa protein indicated by the arrow was HMGB1. C , HMGB1 in cytosolic fraction of HepG2 cells was pulled down with the metformin beads in the presence of indicated concentrations of metformin. D , recombinant HMGB1 was pulled down with the metformin beads or control beads in the presence of the indicated concentrations of metformin, phenformin, putrescine, or 6-aminohexanoic acid. Data shown are representative of three independent experiments with similar results.

    Journal: The Journal of Biological Chemistry

    Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

    doi: 10.1074/jbc.M116.769380

    Figure Lengend Snippet: Identification of HMGB1 as a novel metformin-binding protein. A , structure of metformin ( upper ) and the biotinylated compound containing metformin-like biguanide structure ( lower ) used for the affinity chromatography. B , affinity purification was performed from rat liver cytosol with the metformin beads and the control biotin beads. Bead eluates were analyzed by SDS-PAGE followed by silver staining. Mass spectrometry analysis revealed the ∼25-kDa protein indicated by the arrow was HMGB1. C , HMGB1 in cytosolic fraction of HepG2 cells was pulled down with the metformin beads in the presence of indicated concentrations of metformin. D , recombinant HMGB1 was pulled down with the metformin beads or control beads in the presence of the indicated concentrations of metformin, phenformin, putrescine, or 6-aminohexanoic acid. Data shown are representative of three independent experiments with similar results.

    Article Snippet: Materials and antibodies Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N -[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma.

    Techniques: Binding Assay, Affinity Chromatography, Affinity Purification, SDS Page, Silver Staining, Mass Spectrometry, Recombinant

    Metformin inhibited p38 phosphorylation induced by HMGB1 but not that induced by HMGB1-ΔAT in RAW264.7 cells. RAW264.7 cells were incubated with 1 μg/ml full-length HMGB1, HMGB1-ΔAT, or LPS in the absence or presence of indicated concentrations of metformin at 37 °C for 1 h. HMGB1 or LPS were preincubated without or with metformin at 4 °C for 24 h before the addition to cells. Cell lysates were then analyzed by immunoblotting for phosphorylated and total p38 and for phosphorylated and total AMPK. A , typical photos of immunoblotting. Data shown are representative of four independent experiments with similar results. B and C , ratios of phosphorylated/total p38 ( B ) and phosphorylated/total AMPK ( C ) were calculated from densitometry analysis. B and C , results were analyzed as the percentage of control group (HMGB1 alone in left panels , HMGB1-ΔAT alone in center panels, and LPS alone in left panels ). Data are presented as mean ± S.E. ( n = 4). ***; p

    Journal: The Journal of Biological Chemistry

    Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

    doi: 10.1074/jbc.M116.769380

    Figure Lengend Snippet: Metformin inhibited p38 phosphorylation induced by HMGB1 but not that induced by HMGB1-ΔAT in RAW264.7 cells. RAW264.7 cells were incubated with 1 μg/ml full-length HMGB1, HMGB1-ΔAT, or LPS in the absence or presence of indicated concentrations of metformin at 37 °C for 1 h. HMGB1 or LPS were preincubated without or with metformin at 4 °C for 24 h before the addition to cells. Cell lysates were then analyzed by immunoblotting for phosphorylated and total p38 and for phosphorylated and total AMPK. A , typical photos of immunoblotting. Data shown are representative of four independent experiments with similar results. B and C , ratios of phosphorylated/total p38 ( B ) and phosphorylated/total AMPK ( C ) were calculated from densitometry analysis. B and C , results were analyzed as the percentage of control group (HMGB1 alone in left panels , HMGB1-ΔAT alone in center panels, and LPS alone in left panels ). Data are presented as mean ± S.E. ( n = 4). ***; p

    Article Snippet: Materials and antibodies Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N -[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma.

    Techniques: Incubation

    Metformin inhibited HMGB1-induced p38 phosphorylation in mouse peritoneal macrophages. Mouse peritoneal macrophages from Balb/c mice were incubated at 37 °C for 1 h with 1 μg/ml full-length HMGB1 or 1 μg/ml HMGB1-ΔAT in the absence or presence of 3 m m metformin. HMGB1 or HMGB1-ΔAT was preincubated without or with metformin at 4 °C for 24 h before the addition to cells. Cell lysates were then analyzed by immunoblotting for phosphorylated and total p38 and for phosphorylated and total AMPK. A , typical photos of immunoblots. Data shown are representative of four independent experiments with similar results. B and C , ratios of phosphorylated/total p38 ( B ) and phosphorylated/total AMPK ( C ) were calculated from densitometry analysis. B and C , results were analyzed as the percentage of control group (HMGB1 alone or HMGB1-ΔAT alone). Data are presented as mean ± S.E. ( n = 4). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

    doi: 10.1074/jbc.M116.769380

    Figure Lengend Snippet: Metformin inhibited HMGB1-induced p38 phosphorylation in mouse peritoneal macrophages. Mouse peritoneal macrophages from Balb/c mice were incubated at 37 °C for 1 h with 1 μg/ml full-length HMGB1 or 1 μg/ml HMGB1-ΔAT in the absence or presence of 3 m m metformin. HMGB1 or HMGB1-ΔAT was preincubated without or with metformin at 4 °C for 24 h before the addition to cells. Cell lysates were then analyzed by immunoblotting for phosphorylated and total p38 and for phosphorylated and total AMPK. A , typical photos of immunoblots. Data shown are representative of four independent experiments with similar results. B and C , ratios of phosphorylated/total p38 ( B ) and phosphorylated/total AMPK ( C ) were calculated from densitometry analysis. B and C , results were analyzed as the percentage of control group (HMGB1 alone or HMGB1-ΔAT alone). Data are presented as mean ± S.E. ( n = 4). *, p

    Article Snippet: Materials and antibodies Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N -[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma.

    Techniques: Mouse Assay, Incubation, Western Blot

    Metformin inhibited HMGB1-induced p38 phosphorylation in RAW264.7 cells in a preincubation time-dependent manner. RAW264.7 cells were stimulated with 1 μg/ml HMGB1 in the absence or presence of 10 m m metformin at 37 °C for 1 h. HMGB1 was preincubated with metformin at 4 °C for the indicated periods before the addition to cells. Cell lysates were subsequently analyzed by immunoblotting for phosphorylated and total p38 and phosphorylated and total AMPK. A , typical photos of immunoblots. Data shown are representative of four independent experiments with similar results. B and C , ratios of phosphorylated/total p38 ( B ) and phosphorylated/total AMPK ( C ) were calculated from densitometry analysis. B and C , results were analyzed as the percentage of control group (HMGB1 alone). Data are presented as the mean ± S.E. ( n = 4). **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

    doi: 10.1074/jbc.M116.769380

    Figure Lengend Snippet: Metformin inhibited HMGB1-induced p38 phosphorylation in RAW264.7 cells in a preincubation time-dependent manner. RAW264.7 cells were stimulated with 1 μg/ml HMGB1 in the absence or presence of 10 m m metformin at 37 °C for 1 h. HMGB1 was preincubated with metformin at 4 °C for the indicated periods before the addition to cells. Cell lysates were subsequently analyzed by immunoblotting for phosphorylated and total p38 and phosphorylated and total AMPK. A , typical photos of immunoblots. Data shown are representative of four independent experiments with similar results. B and C , ratios of phosphorylated/total p38 ( B ) and phosphorylated/total AMPK ( C ) were calculated from densitometry analysis. B and C , results were analyzed as the percentage of control group (HMGB1 alone). Data are presented as the mean ± S.E. ( n = 4). **, p

    Article Snippet: Materials and antibodies Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N -[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma.

    Techniques: Western Blot

    Metformin bound to HMGB1 through its acidic tail. A , domain structure of HMGB1 and its mutants used in this study. B , purified His-tagged HMGB1 and its mutants were pulled down with the metformin beads and detected by immunoblotting with anti-His antibody. I , input (30%); C , control beads; M , metformin beads. C , full-length HMGB1 was pulled down with the metformin beads in the presence of the indicated concentrations of the acidic tail or HMGB1-ΔAT. Data shown are representative of three independent experiments with similar results.

    Journal: The Journal of Biological Chemistry

    Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

    doi: 10.1074/jbc.M116.769380

    Figure Lengend Snippet: Metformin bound to HMGB1 through its acidic tail. A , domain structure of HMGB1 and its mutants used in this study. B , purified His-tagged HMGB1 and its mutants were pulled down with the metformin beads and detected by immunoblotting with anti-His antibody. I , input (30%); C , control beads; M , metformin beads. C , full-length HMGB1 was pulled down with the metformin beads in the presence of the indicated concentrations of the acidic tail or HMGB1-ΔAT. Data shown are representative of three independent experiments with similar results.

    Article Snippet: Materials and antibodies Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N -[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma.

    Techniques: Purification

    Metformin inhibited HMGB1-induced TNFα elevation in vivo . A and B , Balb/c mice were intraperitoneally administered full-length HMGB1 or HMGB1-ΔAT at 1 mg/kg ( A ) or LPS at 0.1 mg/kg ( B ) in the absence or presence of 300 mg/kg metformin. As a control, PBS vehicle was injected. In some experiments HMGB1, HMGB1-ΔAT, or LPS was preincubated with metformin at 4 °C for 24 h before injection. Serum TNFα levels were measured by ELISA 2 h after peritoneal administration. Data are presented as the mean ± S.E. ( n = 7). **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

    doi: 10.1074/jbc.M116.769380

    Figure Lengend Snippet: Metformin inhibited HMGB1-induced TNFα elevation in vivo . A and B , Balb/c mice were intraperitoneally administered full-length HMGB1 or HMGB1-ΔAT at 1 mg/kg ( A ) or LPS at 0.1 mg/kg ( B ) in the absence or presence of 300 mg/kg metformin. As a control, PBS vehicle was injected. In some experiments HMGB1, HMGB1-ΔAT, or LPS was preincubated with metformin at 4 °C for 24 h before injection. Serum TNFα levels were measured by ELISA 2 h after peritoneal administration. Data are presented as the mean ± S.E. ( n = 7). **, p

    Article Snippet: Materials and antibodies Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N -[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma.

    Techniques: In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    Metformin did not exhibit further inhibition when extracellular function of HMGB1 was blocked by anti-HMGB1-neutralizing antibody in the acetaminophen-induced liver injury model mice. Balb/c mice were intraperitoneally administered 400 mg/kg acetaminophen without or with 350 mg/kg metformin. Control antibody or anti-HMGB1 antibody at 5 mg/kg was simultaneously administered intravenously. A , serum ALT levels were measured 5 h after acetaminophen administration. Data are presented as the mean ± S.E. ( n = 6–8). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

    doi: 10.1074/jbc.M116.769380

    Figure Lengend Snippet: Metformin did not exhibit further inhibition when extracellular function of HMGB1 was blocked by anti-HMGB1-neutralizing antibody in the acetaminophen-induced liver injury model mice. Balb/c mice were intraperitoneally administered 400 mg/kg acetaminophen without or with 350 mg/kg metformin. Control antibody or anti-HMGB1 antibody at 5 mg/kg was simultaneously administered intravenously. A , serum ALT levels were measured 5 h after acetaminophen administration. Data are presented as the mean ± S.E. ( n = 6–8). *, p

    Article Snippet: Materials and antibodies Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N -[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma.

    Techniques: Inhibition, Mouse Assay

    Metformin did not change the redox states or molecular weight of HMGB1. A , HMGB1 was incubated at 4 °C in the absence or presence of 100 m m metformin for the indicated time periods and analyzed by SDS-PAGE without or with β-mercaptoethanol ( 2-ME ) treatment at 95 °C for 5 min. HMGB1 was detected by immunoblotting with anti-HMGB1 antibody. It is noted that disulfide and fully-reduced HMGB1 can be separated by mobility in SDS-PAGE without 2-ME treatment. Bands in the marker ( M ) lane indicated 30, 40, and 50 kDa from the bottom. B , mass spectrometry analysis of HMGB1 incubated without ( upper diagram ) or with 100 m m metformin ( lower diagram ) at 4 °C for 24 h.

    Journal: The Journal of Biological Chemistry

    Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

    doi: 10.1074/jbc.M116.769380

    Figure Lengend Snippet: Metformin did not change the redox states or molecular weight of HMGB1. A , HMGB1 was incubated at 4 °C in the absence or presence of 100 m m metformin for the indicated time periods and analyzed by SDS-PAGE without or with β-mercaptoethanol ( 2-ME ) treatment at 95 °C for 5 min. HMGB1 was detected by immunoblotting with anti-HMGB1 antibody. It is noted that disulfide and fully-reduced HMGB1 can be separated by mobility in SDS-PAGE without 2-ME treatment. Bands in the marker ( M ) lane indicated 30, 40, and 50 kDa from the bottom. B , mass spectrometry analysis of HMGB1 incubated without ( upper diagram ) or with 100 m m metformin ( lower diagram ) at 4 °C for 24 h.

    Article Snippet: Materials and antibodies Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N -[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma.

    Techniques: Molecular Weight, Incubation, SDS Page, Marker, Mass Spectrometry

    Site-specific incorporation of AzF into recombinant GLP-1 fused to sfGFP. ( A ) Coomassie Brilliant Blue-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) image of cell lysates before (BI) and after (AI) induction; the prominent band attributable to the sfGFP-GLP-1 fusion protein is indicated (arrow). Protein molecular weight standards are shown in lane M. ( B ) Purified sfGFP-GLP1_16AzF on Coomassie Brilliant Blue-stained SDS-PAGE gel after nickel–nitrilotriacetic acid (Ni-NTA) chromatography and anion exchange chromatography (arrow). Protein molecular weight standards are shown in lane M. ( C ) Protein gel image of sfGFP-GLP1_16AzF treated with or without fluorescent dye (DBCO-PEG4-carboxyrhodamine). The gel was subjected to UV irradiation (302 nm) to excite the fluorophore (fluorescence) and stained with Coomassie Brilliant Blue (Coomassie) to visualize the protein.

    Journal: Pharmaceutics

    Article Title: Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide

    doi: 10.3390/pharmaceutics12040364

    Figure Lengend Snippet: Site-specific incorporation of AzF into recombinant GLP-1 fused to sfGFP. ( A ) Coomassie Brilliant Blue-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) image of cell lysates before (BI) and after (AI) induction; the prominent band attributable to the sfGFP-GLP-1 fusion protein is indicated (arrow). Protein molecular weight standards are shown in lane M. ( B ) Purified sfGFP-GLP1_16AzF on Coomassie Brilliant Blue-stained SDS-PAGE gel after nickel–nitrilotriacetic acid (Ni-NTA) chromatography and anion exchange chromatography (arrow). Protein molecular weight standards are shown in lane M. ( C ) Protein gel image of sfGFP-GLP1_16AzF treated with or without fluorescent dye (DBCO-PEG4-carboxyrhodamine). The gel was subjected to UV irradiation (302 nm) to excite the fluorophore (fluorescence) and stained with Coomassie Brilliant Blue (Coomassie) to visualize the protein.

    Article Snippet: Anti-GLP-1 monoclonal antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Recombinant, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Molecular Weight, Purification, Chromatography, Irradiation, Fluorescence