3t3-l1 adipocytes Search Results


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  • 99
    ATCC 3t3l1 pre adipocytes culture 3t3l1 pre adipocytes
    Expression of CRF receptors and peptides in <t>3T3L1</t> pre-adipocytes, differentiated adipocytes and human primary white adipocytes. Panel A, (a), Cells were exposed to antalarmin (a CRF 1 receptor antagonist) and/or astressin-2B (a CRF 2 receptor antagonist) and the decrease of specific binding activity was measured. Data are expressed as the decrease of specific binding (mean±SE, n = 4 of two independent experiments) and *p
    3t3l1 Pre Adipocytes Culture 3t3l1 Pre Adipocytes, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 3t3 l1 adipocytes
    Effects of ROS inhibitors on FFA-induced ER stress in <t>3T3-L1</t> adipocytes. A, Treatment with FFA up-regulated sXbp-1 mRNA in 3T3-L1 adipocytes. <t>3T3-L1</t> adipocytes were treated with 50 μg/ml FFA/BSA for 4 hours. uXbp-1 , unspliced forms of Xbp-1 mRNA; sXbp-1 , spliced forms of Xbp-1 mRNA. B, mRNA expression levels of ER stress markers in 3T3-L1 adipocytes treated with FFA. 3T3-L1 adipocytes were treated with 50 μg/ml FFA/BSA for 4 hours. mRNA expression levels ER stress markers were significantly increased in treatment with FFA. Values are mean ± SD (n = 3). *p
    3t3 L1 Adipocytes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Syntaxin 3t3 l1 adipocytes
    Munc18c residues Tyr219 and Tyr521 are essential for insulin-stimulated GLUT4 vesicle exocytosis. (A) <t>3T3-L1</t> adipocytes coelectroporated with Munc18c-specific (siMunc18c) or nontargeting control (siCon) shRNA plus Myc-GLUT4-GFP plasmid DNAs were left unstimulated or were stimulated with 100 nM insulin for 30 min followed by fixation but not permeabilization. Exofacial Myc exposure was determined by anti-Myc immunostaining (red) followed by immunofluorescent confocal microscopy. GFP fluorescence was examined to indicate subcellular location of GLUT4-GFP vesicles (green). The number of Myc-stained cells relative to the total number of GFP-fluorescing cells (≥50 cells per condition) was quantified in at least three independent sets of cells as depicted in the bar graph inset; *, P
    3t3 L1 Adipocytes, supplied by Syntaxin, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Trinity Biotech 3t3 l1 adipocytes
    Munc18c residues Tyr219 and Tyr521 are essential for insulin-stimulated GLUT4 vesicle exocytosis. (A) <t>3T3-L1</t> adipocytes coelectroporated with Munc18c-specific (siMunc18c) or nontargeting control (siCon) shRNA plus Myc-GLUT4-GFP plasmid DNAs were left unstimulated or were stimulated with 100 nM insulin for 30 min followed by fixation but not permeabilization. Exofacial Myc exposure was determined by anti-Myc immunostaining (red) followed by immunofluorescent confocal microscopy. GFP fluorescence was examined to indicate subcellular location of GLUT4-GFP vesicles (green). The number of Myc-stained cells relative to the total number of GFP-fluorescing cells (≥50 cells per condition) was quantified in at least three independent sets of cells as depicted in the bar graph inset; *, P
    3t3 L1 Adipocytes, supplied by Trinity Biotech, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ZenBio 3t3 l1 pre adipocytes
    Generation of a novel stable <t>3T3-L1</t> adipocyte model of insulin receptoropathy. ( a ) Concatenated miR-shRNAs targeting murine Insr in exon 2 and exon 9 preceded by GFP under the control of a tet-responsive element was packaged into third-generation lentivirus to enable transduction of 3T3-L1 pre-adipocytes. The exploded view shows the nucleotide mismatches between the mouse Insr targeted by each miR-shRNA with the human INSR sequence. Green shaded elements of the transgene are inducible by the addition of DOX. Transduced 3T3-L1 pre-adipocytes underwent single cell clonal selection in the presence of hygromycin to generate 3T3-L1 MmINSRKD. ( b ) 3T3-L1 MmINSRKD cells were then transduced with a second lentivirus encoding C-terminal myc-tagged human INSR transgenes under the control of a tet-responsive element and underwent polyclonal selection in the presence of neomycin to generate 3T3-L1 MmINSRKD hINSR . ( c ) Western blots of whole-cell lysates from day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT cells grown in the presence of increasing concentrations of DOX for 72 h. ( d ) Densitometry analysis of western blots from three independent experiments demonstrating knockdown of endogenous mouse Insr and expression of human INSR with increasing concentrations of DOX. ( e ) Western blots of whole-cell lysates from day 16 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR (mutant INSR as indicated) cells grown in the presence of 1 μg/ml DOX for 10 days. ( f ) Oil Red O staining of lipid accumulation in day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT or mutant (as indicated) cells grown ± DOX (1 μg/ml) for 72 h. GFP, green fluorescent protein; Hygro, hygromycin resistance; IRES, internal ribosome entry site; Mm, murine; Neo, neomycin resistance; rtTA3, reverse tetracycline-controlled transactivator; TRE, tet-response element; Ubi-C, ubiquitin C promoter
    3t3 L1 Pre Adipocytes, supplied by ZenBio, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ZenBio 3t3 l1 adipocyte kit
    Generation of a novel stable <t>3T3-L1</t> adipocyte model of insulin receptoropathy. ( a ) Concatenated miR-shRNAs targeting murine Insr in exon 2 and exon 9 preceded by GFP under the control of a tet-responsive element was packaged into third-generation lentivirus to enable transduction of 3T3-L1 pre-adipocytes. The exploded view shows the nucleotide mismatches between the mouse Insr targeted by each miR-shRNA with the human INSR sequence. Green shaded elements of the transgene are inducible by the addition of DOX. Transduced 3T3-L1 pre-adipocytes underwent single cell clonal selection in the presence of hygromycin to generate 3T3-L1 MmINSRKD. ( b ) 3T3-L1 MmINSRKD cells were then transduced with a second lentivirus encoding C-terminal myc-tagged human INSR transgenes under the control of a tet-responsive element and underwent polyclonal selection in the presence of neomycin to generate 3T3-L1 MmINSRKD hINSR . ( c ) Western blots of whole-cell lysates from day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT cells grown in the presence of increasing concentrations of DOX for 72 h. ( d ) Densitometry analysis of western blots from three independent experiments demonstrating knockdown of endogenous mouse Insr and expression of human INSR with increasing concentrations of DOX. ( e ) Western blots of whole-cell lysates from day 16 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR (mutant INSR as indicated) cells grown in the presence of 1 μg/ml DOX for 10 days. ( f ) Oil Red O staining of lipid accumulation in day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT or mutant (as indicated) cells grown ± DOX (1 μg/ml) for 72 h. GFP, green fluorescent protein; Hygro, hygromycin resistance; IRES, internal ribosome entry site; Mm, murine; Neo, neomycin resistance; rtTA3, reverse tetracycline-controlled transactivator; TRE, tet-response element; Ubi-C, ubiquitin C promoter
    3t3 L1 Adipocyte Kit, supplied by ZenBio, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ZenBio 3t3 l1 adipocyte medium
    Generation of a novel stable <t>3T3-L1</t> adipocyte model of insulin receptoropathy. ( a ) Concatenated miR-shRNAs targeting murine Insr in exon 2 and exon 9 preceded by GFP under the control of a tet-responsive element was packaged into third-generation lentivirus to enable transduction of 3T3-L1 pre-adipocytes. The exploded view shows the nucleotide mismatches between the mouse Insr targeted by each miR-shRNA with the human INSR sequence. Green shaded elements of the transgene are inducible by the addition of DOX. Transduced 3T3-L1 pre-adipocytes underwent single cell clonal selection in the presence of hygromycin to generate 3T3-L1 MmINSRKD. ( b ) 3T3-L1 MmINSRKD cells were then transduced with a second lentivirus encoding C-terminal myc-tagged human INSR transgenes under the control of a tet-responsive element and underwent polyclonal selection in the presence of neomycin to generate 3T3-L1 MmINSRKD hINSR . ( c ) Western blots of whole-cell lysates from day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT cells grown in the presence of increasing concentrations of DOX for 72 h. ( d ) Densitometry analysis of western blots from three independent experiments demonstrating knockdown of endogenous mouse Insr and expression of human INSR with increasing concentrations of DOX. ( e ) Western blots of whole-cell lysates from day 16 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR (mutant INSR as indicated) cells grown in the presence of 1 μg/ml DOX for 10 days. ( f ) Oil Red O staining of lipid accumulation in day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT or mutant (as indicated) cells grown ± DOX (1 μg/ml) for 72 h. GFP, green fluorescent protein; Hygro, hygromycin resistance; IRES, internal ribosome entry site; Mm, murine; Neo, neomycin resistance; rtTA3, reverse tetracycline-controlled transactivator; TRE, tet-response element; Ubi-C, ubiquitin C promoter
    3t3 L1 Adipocyte Medium, supplied by ZenBio, used in various techniques. Bioz Stars score: 85/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore differentiated 3t3 l1 adipocytes 3t3 l1 preadipocytes
    Generation of a novel stable <t>3T3-L1</t> adipocyte model of insulin receptoropathy. ( a ) Concatenated miR-shRNAs targeting murine Insr in exon 2 and exon 9 preceded by GFP under the control of a tet-responsive element was packaged into third-generation lentivirus to enable transduction of 3T3-L1 pre-adipocytes. The exploded view shows the nucleotide mismatches between the mouse Insr targeted by each miR-shRNA with the human INSR sequence. Green shaded elements of the transgene are inducible by the addition of DOX. Transduced 3T3-L1 pre-adipocytes underwent single cell clonal selection in the presence of hygromycin to generate 3T3-L1 MmINSRKD. ( b ) 3T3-L1 MmINSRKD cells were then transduced with a second lentivirus encoding C-terminal myc-tagged human INSR transgenes under the control of a tet-responsive element and underwent polyclonal selection in the presence of neomycin to generate 3T3-L1 MmINSRKD hINSR . ( c ) Western blots of whole-cell lysates from day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT cells grown in the presence of increasing concentrations of DOX for 72 h. ( d ) Densitometry analysis of western blots from three independent experiments demonstrating knockdown of endogenous mouse Insr and expression of human INSR with increasing concentrations of DOX. ( e ) Western blots of whole-cell lysates from day 16 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR (mutant INSR as indicated) cells grown in the presence of 1 μg/ml DOX for 10 days. ( f ) Oil Red O staining of lipid accumulation in day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT or mutant (as indicated) cells grown ± DOX (1 μg/ml) for 72 h. GFP, green fluorescent protein; Hygro, hygromycin resistance; IRES, internal ribosome entry site; Mm, murine; Neo, neomycin resistance; rtTA3, reverse tetracycline-controlled transactivator; TRE, tet-response element; Ubi-C, ubiquitin C promoter
    Differentiated 3t3 L1 Adipocytes 3t3 L1 Preadipocytes, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DS Pharma Biomedical 3t3 l1 adipocyte medium
    Generation of a novel stable <t>3T3-L1</t> adipocyte model of insulin receptoropathy. ( a ) Concatenated miR-shRNAs targeting murine Insr in exon 2 and exon 9 preceded by GFP under the control of a tet-responsive element was packaged into third-generation lentivirus to enable transduction of 3T3-L1 pre-adipocytes. The exploded view shows the nucleotide mismatches between the mouse Insr targeted by each miR-shRNA with the human INSR sequence. Green shaded elements of the transgene are inducible by the addition of DOX. Transduced 3T3-L1 pre-adipocytes underwent single cell clonal selection in the presence of hygromycin to generate 3T3-L1 MmINSRKD. ( b ) 3T3-L1 MmINSRKD cells were then transduced with a second lentivirus encoding C-terminal myc-tagged human INSR transgenes under the control of a tet-responsive element and underwent polyclonal selection in the presence of neomycin to generate 3T3-L1 MmINSRKD hINSR . ( c ) Western blots of whole-cell lysates from day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT cells grown in the presence of increasing concentrations of DOX for 72 h. ( d ) Densitometry analysis of western blots from three independent experiments demonstrating knockdown of endogenous mouse Insr and expression of human INSR with increasing concentrations of DOX. ( e ) Western blots of whole-cell lysates from day 16 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR (mutant INSR as indicated) cells grown in the presence of 1 μg/ml DOX for 10 days. ( f ) Oil Red O staining of lipid accumulation in day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT or mutant (as indicated) cells grown ± DOX (1 μg/ml) for 72 h. GFP, green fluorescent protein; Hygro, hygromycin resistance; IRES, internal ribosome entry site; Mm, murine; Neo, neomycin resistance; rtTA3, reverse tetracycline-controlled transactivator; TRE, tet-response element; Ubi-C, ubiquitin C promoter
    3t3 L1 Adipocyte Medium, supplied by DS Pharma Biomedical, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ZenBio murine 3t3 l1 pre adipocytes
    Generation of a novel stable <t>3T3-L1</t> adipocyte model of insulin receptoropathy. ( a ) Concatenated miR-shRNAs targeting murine Insr in exon 2 and exon 9 preceded by GFP under the control of a tet-responsive element was packaged into third-generation lentivirus to enable transduction of 3T3-L1 pre-adipocytes. The exploded view shows the nucleotide mismatches between the mouse Insr targeted by each miR-shRNA with the human INSR sequence. Green shaded elements of the transgene are inducible by the addition of DOX. Transduced 3T3-L1 pre-adipocytes underwent single cell clonal selection in the presence of hygromycin to generate 3T3-L1 MmINSRKD. ( b ) 3T3-L1 MmINSRKD cells were then transduced with a second lentivirus encoding C-terminal myc-tagged human INSR transgenes under the control of a tet-responsive element and underwent polyclonal selection in the presence of neomycin to generate 3T3-L1 MmINSRKD hINSR . ( c ) Western blots of whole-cell lysates from day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT cells grown in the presence of increasing concentrations of DOX for 72 h. ( d ) Densitometry analysis of western blots from three independent experiments demonstrating knockdown of endogenous mouse Insr and expression of human INSR with increasing concentrations of DOX. ( e ) Western blots of whole-cell lysates from day 16 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR (mutant INSR as indicated) cells grown in the presence of 1 μg/ml DOX for 10 days. ( f ) Oil Red O staining of lipid accumulation in day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT or mutant (as indicated) cells grown ± DOX (1 μg/ml) for 72 h. GFP, green fluorescent protein; Hygro, hygromycin resistance; IRES, internal ribosome entry site; Mm, murine; Neo, neomycin resistance; rtTA3, reverse tetracycline-controlled transactivator; TRE, tet-response element; Ubi-C, ubiquitin C promoter
    Murine 3t3 L1 Pre Adipocytes, supplied by ZenBio, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Amaxa 3t3 l1 adipocytes
    PTN and PTHR role in mammary epithelial cell migration A) Mammary epithelial cells, HC-11 and differentiated <t>3T3-L1</t> adipocytes were lysed and tested for the presence of PTN, and its receptor PTPRζ1 by Western blotting. Left panel: PTN is highly expressed (upper panel) by 3T3-L1 adipocytes, it is undetectable in HC-11 cells; PTN-receptor PTPRζ1 is highly expressed in HC-11 cells but minimally expressed in the 3T3-L1 adipocytes (middle left panel). Both tubulin and vinculin serve as loading control because of difference in their expression between the cell types. Right panel: differentiated cell lysates (1/5 of total) and serum-free conditioned medium (1/100 of total), collected after 48hrs of incubation, tested for PTN by Western blotting. B) PTN was knocked down in differentiated 3T3-L1 adipocytes by pooled siRNA and C) PTHR was knocked down in 3T3-L1 using pooled PTHR-siRNA as described in Methods (middle panel). The treatments impaired cell migration of HC-11 cells in Boyden chambers (upper panels) (p
    3t3 L1 Adipocytes, supplied by Amaxa, used in various techniques. Bioz Stars score: 92/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific 3t3 l1 adipocytes
    The expression and localization of the exocyst complex in adipocytes. (A) The mRNA expression of Exo70, Sec6, and Sec8 before and after <t>3T3-L1</t> adipocyte differentiation. Pread: 3T1-L1 preadipocytes, Ad: 3T3-L1 adipocytes. (B) Upper, intracellular localization of mCherry-Exo70 (red) and lipid droplets stained with BODIPY 493/503 (green); lower, HA-Sec8 (red) and lipid droplets (green). Scale = 10 μm.
    3t3 L1 Adipocytes, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 3t3 l1 adipocytes
    The expression and localization of the exocyst complex in adipocytes. (A) The mRNA expression of Exo70, Sec6, and Sec8 before and after <t>3T3-L1</t> adipocyte differentiation. Pread: 3T1-L1 preadipocytes, Ad: 3T3-L1 adipocytes. (B) Upper, intracellular localization of mCherry-Exo70 (red) and lipid droplets stained with BODIPY 493/503 (green); lower, HA-Sec8 (red) and lipid droplets (green). Scale = 10 μm.
    3t3 L1 Adipocytes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 891 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Metabolon Inc 3t3 l1 adipocytes
    Ceramide and triacylglycerol levels in treated <t>3T3-L1</t> adipocytes. (A) Ceramide levels were measured with a monoclonal antibody assay in extracts from adipocytes 24 h after addition of 50 μM (gray bars) or 100 μM (white bars) LA or t 10 c 12 CLA (CLA) with or without 30μM C2 ceramide (C2) or 10μg/ml of an inhibitor of sphingosine kinase (SKI-II), which is expected to increase ceramide levels. (B) Triacylglycerol (TG) levels 24 h after <t>3T3-L1</t> adipocytes were treated with 50 μM LA or t 10 c 12 CLA (CLA), with or without 30 μM C2 ceramide or 10 μg/ml SKI-II. The control sample contained 0.2% DMSO, which produced the same values as a control sample containing 0.2% ethanol in separate experiments (data not shown). (C) Triacylglycerol levels 24 h after 3T3-L1 adipocytes were treated 100 μM LA or t 10 c 12 CLA (CLA), with or without 20 μM fumonisin B1 (FB1) or 50 μM myriocin (Myri.). (D) Ceramide levels were detected and quantitated as in A for adipocytes treated for 24 h with 100 μM LA or t 10 c 12 CLA, with or without 20 μM FB1 or 50 μM myriocin. Each bar represents the mean ± SEM (n = 3), and a representative experiment of at least three independent experiments is shown. Means not sharing a common letter differ, p ≤0.05.
    3t3 L1 Adipocytes, supplied by Metabolon Inc, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche 3t3 l1 adipocytes
    Treatment of <t>3T3-L1</t> adipocytes with BoNT D blocks insulin-stimulated GLUT4–vesicle translocation to the plasma membrane. ( A ) SLO-permeabilized 3T3-L1 adipocytes were pretreated for 20 min with buffer alone (control) or buffer containing 100 nM BoNT D or BoNT C, followed by an additional 10-min incubation with insulin. Plasma membrane sheets were then prepared and GLUT4 was detected by immunofluorescence. ( B ) Following treatment of permeabilized cells with buffer alone (−) or BoNT D (+), an LDM subcellular membrane fraction was prepared and LDM-resident proteins were examined by immunoblot analysis using antisera against the proteins indicated.
    3t3 L1 Adipocytes, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore adipocyte differentiated 3t3 l1 cells
    Adipocyte-derived molecules that support eosinophil migration and survival account for eosinophil infiltration in the perigonadal adipose tissue of high fat diet (HFD) fed mice. ( a ) mRNA expression of CC chemokine ligand 11 ( Ccl11 ) and Ccl24 in adipocyte-differentiated <t>3T3-L1</t> cells. Representative blots from 3 independent experiments. The full-length blots are presented in Fig. S6 . *** p
    Adipocyte Differentiated 3t3 L1 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of CRF receptors and peptides in 3T3L1 pre-adipocytes, differentiated adipocytes and human primary white adipocytes. Panel A, (a), Cells were exposed to antalarmin (a CRF 1 receptor antagonist) and/or astressin-2B (a CRF 2 receptor antagonist) and the decrease of specific binding activity was measured. Data are expressed as the decrease of specific binding (mean±SE, n = 4 of two independent experiments) and *p

    Journal: PLoS ONE

    Article Title: Corticotrophin-Releasing Factor (CRF) and the Urocortins Are Potent Regulators of the Inflammatory Phenotype of Human and Mouse White Adipocytes and the Differentiation of Mouse 3T3L1 Pre-Adipocytes

    doi: 10.1371/journal.pone.0097060

    Figure Lengend Snippet: Expression of CRF receptors and peptides in 3T3L1 pre-adipocytes, differentiated adipocytes and human primary white adipocytes. Panel A, (a), Cells were exposed to antalarmin (a CRF 1 receptor antagonist) and/or astressin-2B (a CRF 2 receptor antagonist) and the decrease of specific binding activity was measured. Data are expressed as the decrease of specific binding (mean±SE, n = 4 of two independent experiments) and *p

    Article Snippet: 3T3L1 Pre-adipocytes Culture 3T3L1 pre-adipocytes were obtained from the American Type Culture Collection and cultured in a basal medium (DMEM, 4 mM L-Glutamine, 25 mM D-Glucose, 1 mM Sodium Pyruvate, 3.7 g/L NaHCO3 , 50 units/ml penicillin, and 50 µg/ml streptomycin) supplemented with 10% NBS (growth medium) in a humidified atmosphere at 37 C. The medium was changed three times a week.

    Techniques: Expressing, Binding Assay, Activity Assay

    Effect of CRF agonists on the differentiation process of 3T3L1 cells. Pre-adipocytes were exposed to CRF peptides and/or LPS during the differentiation period and stained with Oil-Red-O. Panel A, The accumulation of lipid droplets were visualized under a microscope; Panels B, C and D, The degree of differentiation was measured as the intensity of the absorbance read at 595 nm. Panel E, The accumulation of lipid droplets of cells pre-exposed to antalarmin (10 −6 M) and to CRF or Cortagine at 10 −8 M was measured as the intensity of the absorbance read at 595 nm. Data are expressed as percentage change compared with control values (Panels B, C, E) or compared with LPS (Panel D) (mean±SE, n = 8 of three independent experiments). *p

    Journal: PLoS ONE

    Article Title: Corticotrophin-Releasing Factor (CRF) and the Urocortins Are Potent Regulators of the Inflammatory Phenotype of Human and Mouse White Adipocytes and the Differentiation of Mouse 3T3L1 Pre-Adipocytes

    doi: 10.1371/journal.pone.0097060

    Figure Lengend Snippet: Effect of CRF agonists on the differentiation process of 3T3L1 cells. Pre-adipocytes were exposed to CRF peptides and/or LPS during the differentiation period and stained with Oil-Red-O. Panel A, The accumulation of lipid droplets were visualized under a microscope; Panels B, C and D, The degree of differentiation was measured as the intensity of the absorbance read at 595 nm. Panel E, The accumulation of lipid droplets of cells pre-exposed to antalarmin (10 −6 M) and to CRF or Cortagine at 10 −8 M was measured as the intensity of the absorbance read at 595 nm. Data are expressed as percentage change compared with control values (Panels B, C, E) or compared with LPS (Panel D) (mean±SE, n = 8 of three independent experiments). *p

    Article Snippet: 3T3L1 Pre-adipocytes Culture 3T3L1 pre-adipocytes were obtained from the American Type Culture Collection and cultured in a basal medium (DMEM, 4 mM L-Glutamine, 25 mM D-Glucose, 1 mM Sodium Pyruvate, 3.7 g/L NaHCO3 , 50 units/ml penicillin, and 50 µg/ml streptomycin) supplemented with 10% NBS (growth medium) in a humidified atmosphere at 37 C. The medium was changed three times a week.

    Techniques: Staining, Microscopy

    Effect of CRF on TLR4 and interleukins during differentiation of 3T3L1. Pre-adipocytes were cultured in differentiating media supplemented with CRF at 10 −8 M, pre-treated with antalarmin at 10 −6 M and the production of IL-6 and CXCL1 was measured by ELISA (Panels A, B). Pre-adipocytes were cultured in differentiating media supplemented with CRF at 10 −8 M plus/minus LPS (10 ng/ml) and the expression of TLR4 was measured by RT-PCR (Panel C) and the production of IL-6 and CXCL1 (Panels D, E) was measured by ELISA. Data are expressed as percentage change compared with control values (mean±SE, n = 10 of five independent experiments). *p

    Journal: PLoS ONE

    Article Title: Corticotrophin-Releasing Factor (CRF) and the Urocortins Are Potent Regulators of the Inflammatory Phenotype of Human and Mouse White Adipocytes and the Differentiation of Mouse 3T3L1 Pre-Adipocytes

    doi: 10.1371/journal.pone.0097060

    Figure Lengend Snippet: Effect of CRF on TLR4 and interleukins during differentiation of 3T3L1. Pre-adipocytes were cultured in differentiating media supplemented with CRF at 10 −8 M, pre-treated with antalarmin at 10 −6 M and the production of IL-6 and CXCL1 was measured by ELISA (Panels A, B). Pre-adipocytes were cultured in differentiating media supplemented with CRF at 10 −8 M plus/minus LPS (10 ng/ml) and the expression of TLR4 was measured by RT-PCR (Panel C) and the production of IL-6 and CXCL1 (Panels D, E) was measured by ELISA. Data are expressed as percentage change compared with control values (mean±SE, n = 10 of five independent experiments). *p

    Article Snippet: 3T3L1 Pre-adipocytes Culture 3T3L1 pre-adipocytes were obtained from the American Type Culture Collection and cultured in a basal medium (DMEM, 4 mM L-Glutamine, 25 mM D-Glucose, 1 mM Sodium Pyruvate, 3.7 g/L NaHCO3 , 50 units/ml penicillin, and 50 µg/ml streptomycin) supplemented with 10% NBS (growth medium) in a humidified atmosphere at 37 C. The medium was changed three times a week.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    Newly synthesized IRAP-TfR-AA 76,77 defaults to the plasma membrane, where it accumulates under steady-state conditions. (A) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of IRAP-TfR- WT (panels a and c) or IRAP-TfR -AA 76,77 (panels b and d) and, 16 hours later, cells were serum-starved and treated without (panels a and b) or with (panels c and d) 100 nM insulin for 20 minutes. The cells were then fixed, permeabilized and labeled with an anti-TfR antibody followed by Texas-Red-conjugated secondary antibody, and were then visualized with a Zeiss LSM510 scanning laser confocal microscope. (B) The data in A were quantified as the ratio of surface fluorescence:total fluorescence, as described in the Materials and Methods. White bars, basal cells; black bars, insulin-stimulated cells. (C) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of IRAP-TfR -AA 76,77 and, 16 hours later, cells were incubated with vehicle alone (panels a–d) or vehicle plus 10 μg/ml cycloheximide (CHX) (panels e–h) for 0, 1, 2 or 4 hours. The cells were then processed for confocal microscopy as indicated in A. (D) The data presented in C were quantified by calculating the ratio of plasma membrane:total fluorescence, as described under Materials and Methods. Results are from three independent experiments, with fifteen cells per experiment (mean ± s.e.m.). (E) Fully differentiated 3T3L1 adipocytes were transfected with 50 μg IRAP-TfR -WT or 50 μg IRAP-TfR -AA 76,77 cDNA. After an overnight recovery period, cells were incubated for 0, 0.5, 1, 2, 4 or 6 hours in 10 μg/ml CHX. In lane 1, cells were plated immediately in CHX after electroporation and were then incubated for 6 hours. At the indicated time points, cell lysates were prepared and subjected to western blotting. An anti-IRAP antibody was used to detect both endogenous IRAP (upper panel) and the expressed IRAP-TfR chimera (lower panel). a.u., arbitrary units.

    Journal: Journal of cell science

    Article Title: Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

    doi: 10.1242/jcs.017517

    Figure Lengend Snippet: Newly synthesized IRAP-TfR-AA 76,77 defaults to the plasma membrane, where it accumulates under steady-state conditions. (A) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of IRAP-TfR- WT (panels a and c) or IRAP-TfR -AA 76,77 (panels b and d) and, 16 hours later, cells were serum-starved and treated without (panels a and b) or with (panels c and d) 100 nM insulin for 20 minutes. The cells were then fixed, permeabilized and labeled with an anti-TfR antibody followed by Texas-Red-conjugated secondary antibody, and were then visualized with a Zeiss LSM510 scanning laser confocal microscope. (B) The data in A were quantified as the ratio of surface fluorescence:total fluorescence, as described in the Materials and Methods. White bars, basal cells; black bars, insulin-stimulated cells. (C) Differentiated 3T3L1 adipocytes were electroporated with 50 μg of IRAP-TfR -AA 76,77 and, 16 hours later, cells were incubated with vehicle alone (panels a–d) or vehicle plus 10 μg/ml cycloheximide (CHX) (panels e–h) for 0, 1, 2 or 4 hours. The cells were then processed for confocal microscopy as indicated in A. (D) The data presented in C were quantified by calculating the ratio of plasma membrane:total fluorescence, as described under Materials and Methods. Results are from three independent experiments, with fifteen cells per experiment (mean ± s.e.m.). (E) Fully differentiated 3T3L1 adipocytes were transfected with 50 μg IRAP-TfR -WT or 50 μg IRAP-TfR -AA 76,77 cDNA. After an overnight recovery period, cells were incubated for 0, 0.5, 1, 2, 4 or 6 hours in 10 μg/ml CHX. In lane 1, cells were plated immediately in CHX after electroporation and were then incubated for 6 hours. At the indicated time points, cell lysates were prepared and subjected to western blotting. An anti-IRAP antibody was used to detect both endogenous IRAP (upper panel) and the expressed IRAP-TfR chimera (lower panel). a.u., arbitrary units.

    Article Snippet: 3T3L1 pre-adipocytes were purchased from the American Type Culture Collection, and were cultured and differentiated as described previously ( ).

    Techniques: Synthesized, Labeling, Microscopy, Fluorescence, Incubation, Confocal Microscopy, Transfection, Electroporation, Western Blot

    IRAP-TfR-AA 76,77 displays a normal rate and extent of plasma membrane endocytosis. Differentiated 3T3L1 adipocytes were transfected with 50 μg of IRAP-TfR -WT or IRAP-TfR -AA 76,77 and, 16 hours later, stimulated with 100 nM insulin (this is only necessary to induce TfR-IRAP-WT translocation but was done for both constructs for internal consistency). The cells were then cooled to 4°C and the cell surface was labeled with the anti-TfR antibody. The cells were washed and warmed to 37°C for various times, as indicated. The cells were then fixed, permeabilized and labeled with Texas-Red-conjugated anti-TfR antibody. (A) Representative cell images are shown. (B) The percentage of cell-surface signal was calculated using the Zeiss LSM 510 software package (mean ± s.e.m. of three independent experiments). (C) Fully differentiated 3T3L1 adipocytes were co-electroporated with 50 μg of both HA-GLUT4 and IRAP-TfR- AA 76,77 , and were allowed to recover overnight. Cells were then stimulated with 100 nM insulin for 20 minutes, and then surface-labeled on ice with a mouse monoclonal anti-TfR antibody and a polyclonal rabbit HA antibody for 60 minutes. After extensive washing with ice-cold PBS, cells were either fixed in paraformaldehyde ( t =0) or were warmed to 37°C for 6 hours ( t =6 h), fixed and labeled with Texas-Red-conjugated donkey anti-mouse and Alexa-Fluor-488-conjugated donkey anti-rabbit secondary antibodies. Images were collected on a Zeiss LSM 510 META confocal microscope. a.u., arbitrary units.

    Journal: Journal of cell science

    Article Title: Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

    doi: 10.1242/jcs.017517

    Figure Lengend Snippet: IRAP-TfR-AA 76,77 displays a normal rate and extent of plasma membrane endocytosis. Differentiated 3T3L1 adipocytes were transfected with 50 μg of IRAP-TfR -WT or IRAP-TfR -AA 76,77 and, 16 hours later, stimulated with 100 nM insulin (this is only necessary to induce TfR-IRAP-WT translocation but was done for both constructs for internal consistency). The cells were then cooled to 4°C and the cell surface was labeled with the anti-TfR antibody. The cells were washed and warmed to 37°C for various times, as indicated. The cells were then fixed, permeabilized and labeled with Texas-Red-conjugated anti-TfR antibody. (A) Representative cell images are shown. (B) The percentage of cell-surface signal was calculated using the Zeiss LSM 510 software package (mean ± s.e.m. of three independent experiments). (C) Fully differentiated 3T3L1 adipocytes were co-electroporated with 50 μg of both HA-GLUT4 and IRAP-TfR- AA 76,77 , and were allowed to recover overnight. Cells were then stimulated with 100 nM insulin for 20 minutes, and then surface-labeled on ice with a mouse monoclonal anti-TfR antibody and a polyclonal rabbit HA antibody for 60 minutes. After extensive washing with ice-cold PBS, cells were either fixed in paraformaldehyde ( t =0) or were warmed to 37°C for 6 hours ( t =6 h), fixed and labeled with Texas-Red-conjugated donkey anti-mouse and Alexa-Fluor-488-conjugated donkey anti-rabbit secondary antibodies. Images were collected on a Zeiss LSM 510 META confocal microscope. a.u., arbitrary units.

    Article Snippet: 3T3L1 pre-adipocytes were purchased from the American Type Culture Collection, and were cultured and differentiated as described previously ( ).

    Techniques: Transfection, Translocation Assay, Construct, Labeling, Software, Microscopy

    IRAP-TfR-WT and IRAP-TfR-AA 76,77 both recycle from the cell surface back to an IRC that is BFA-insensitive. (A) Fully differentiated 3T3L1 adipocytes were transfected with 50 μg of either IRAP-TfR -WT or IRAP-TfR -AA 76,77 and, 16 hours later, cells were stimulated with insulin and surface-labeled with the anti-TfR antibody on ice for 60 minutes. Cells were then washed with PBS and incubated at 37°C for 4 hours. For the BFA treatment, cells were incubated with BFA (5 μg/ml) during the last 30 minutes of the washout period and also during the second round of insulin treatment. Control cells were incubated with vehicle alone. Following the second round of treatment without or with insulin (100 nM, 30 minutes), cells were fixed, permeabilized and labeled with Texas-Red-conjugated secondary antibody, as described under Materials and Methods. The ratio of plasma membrane fluorescence:total fluorescence was determined using the Zeiss LSM software package (mean ± s.e.m. of three independent experiments). (B) Control experiments demonstrating the efficacy of 5 μg/ml BFA. Cells were transfected with 50 μg of GFP-IRAP -AA 76,77 reporter construct and immediately plated in media either without (panels a-c) or with (panels d–f) 5 μg/ml BFA. After a 3-hour recovery period, cells were fixed and labeled with an anti-p115 monoclonal antibody, followed by Texas Red secondary antibody. a.u., arbitrary units.

    Journal: Journal of cell science

    Article Title: Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

    doi: 10.1242/jcs.017517

    Figure Lengend Snippet: IRAP-TfR-WT and IRAP-TfR-AA 76,77 both recycle from the cell surface back to an IRC that is BFA-insensitive. (A) Fully differentiated 3T3L1 adipocytes were transfected with 50 μg of either IRAP-TfR -WT or IRAP-TfR -AA 76,77 and, 16 hours later, cells were stimulated with insulin and surface-labeled with the anti-TfR antibody on ice for 60 minutes. Cells were then washed with PBS and incubated at 37°C for 4 hours. For the BFA treatment, cells were incubated with BFA (5 μg/ml) during the last 30 minutes of the washout period and also during the second round of insulin treatment. Control cells were incubated with vehicle alone. Following the second round of treatment without or with insulin (100 nM, 30 minutes), cells were fixed, permeabilized and labeled with Texas-Red-conjugated secondary antibody, as described under Materials and Methods. The ratio of plasma membrane fluorescence:total fluorescence was determined using the Zeiss LSM software package (mean ± s.e.m. of three independent experiments). (B) Control experiments demonstrating the efficacy of 5 μg/ml BFA. Cells were transfected with 50 μg of GFP-IRAP -AA 76,77 reporter construct and immediately plated in media either without (panels a-c) or with (panels d–f) 5 μg/ml BFA. After a 3-hour recovery period, cells were fixed and labeled with an anti-p115 monoclonal antibody, followed by Texas Red secondary antibody. a.u., arbitrary units.

    Article Snippet: 3T3L1 pre-adipocytes were purchased from the American Type Culture Collection, and were cultured and differentiated as described previously ( ).

    Techniques: Transfection, Labeling, Incubation, Fluorescence, Software, Construct

    A dominant-interfering mutant form of AS160 (AS160-4P) inhibits the insulin-stimulated exit of IRAP-TfR-AA 76,77 from the IRC following endocytosis from the cell surface. Fully differentiated 3T3L1 adipocytes were electroporated with 50 μg of IRAP-TfR -AA 76,77 and either AS160 -WT (white bars), or AS160 -4P (black bars). Following a 16-hour recovery period, cells were surface-labeled with the anti-TfR antibody on ice for 60 minutes. Cells were then warmed to 37°C for 6 hours to allow endocytosis to occur, then treated without or with 100 nM insulin for 30 minutes, fixed, permeabilized and labeled with Texas-Red-conjugated secondary antibody as described under Materials and Methods. The ratio of plasma membrane fluorescence to total fluorescence was determined using the Zeiss LSM software package (mean ± s.e.m. of three independent experiments). a.u., arbitrary units.

    Journal: Journal of cell science

    Article Title: Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

    doi: 10.1242/jcs.017517

    Figure Lengend Snippet: A dominant-interfering mutant form of AS160 (AS160-4P) inhibits the insulin-stimulated exit of IRAP-TfR-AA 76,77 from the IRC following endocytosis from the cell surface. Fully differentiated 3T3L1 adipocytes were electroporated with 50 μg of IRAP-TfR -AA 76,77 and either AS160 -WT (white bars), or AS160 -4P (black bars). Following a 16-hour recovery period, cells were surface-labeled with the anti-TfR antibody on ice for 60 minutes. Cells were then warmed to 37°C for 6 hours to allow endocytosis to occur, then treated without or with 100 nM insulin for 30 minutes, fixed, permeabilized and labeled with Texas-Red-conjugated secondary antibody as described under Materials and Methods. The ratio of plasma membrane fluorescence to total fluorescence was determined using the Zeiss LSM software package (mean ± s.e.m. of three independent experiments). a.u., arbitrary units.

    Article Snippet: 3T3L1 pre-adipocytes were purchased from the American Type Culture Collection, and were cultured and differentiated as described previously ( ).

    Techniques: Mutagenesis, Labeling, Fluorescence, Software

    Stx6 function is not required for the insulin-stimulated exit of IRAP from the IRC. (A) Fully differentiated 3T3L1 adipocytes were transfected with 5 nmol of scrambled or Stx6 siRNA and, 72 hours later, were transfected with 50 μg EGFP-IRAP-TfR -WT reporter. Following a 24-hour recovery period, cells were treated with or without insulin, fixed and labeled with Texas Red secondary antibody. The ratio of plasma membrane to total fluorescence was determined using Zeiss LSM software. (B) Cells were transfected with either 5 nmol of scrambled or Stx6 siRNA and, 72 hours later, were transfected with IRAP-TfR -WT reporter. Following an 18-hour recovery period, cells were stimulated with insulin and surface-labeled on ice with anti-TfR monoclonal antibody. Following a 6-hour washout at 37°C, cells were re-stimulated with or without insulin, fixed, labeled with Texas Red secondary antibody and processed for confocal microscopy (mean ± s.e.m. of three independent experiments). A timeline for the transfections is shown above the bar graphs for clarity. Scram, scrambled siRNA. a.u., arbitrary units.

    Journal: Journal of cell science

    Article Title: Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

    doi: 10.1242/jcs.017517

    Figure Lengend Snippet: Stx6 function is not required for the insulin-stimulated exit of IRAP from the IRC. (A) Fully differentiated 3T3L1 adipocytes were transfected with 5 nmol of scrambled or Stx6 siRNA and, 72 hours later, were transfected with 50 μg EGFP-IRAP-TfR -WT reporter. Following a 24-hour recovery period, cells were treated with or without insulin, fixed and labeled with Texas Red secondary antibody. The ratio of plasma membrane to total fluorescence was determined using Zeiss LSM software. (B) Cells were transfected with either 5 nmol of scrambled or Stx6 siRNA and, 72 hours later, were transfected with IRAP-TfR -WT reporter. Following an 18-hour recovery period, cells were stimulated with insulin and surface-labeled on ice with anti-TfR monoclonal antibody. Following a 6-hour washout at 37°C, cells were re-stimulated with or without insulin, fixed, labeled with Texas Red secondary antibody and processed for confocal microscopy (mean ± s.e.m. of three independent experiments). A timeline for the transfections is shown above the bar graphs for clarity. Scram, scrambled siRNA. a.u., arbitrary units.

    Article Snippet: 3T3L1 pre-adipocytes were purchased from the American Type Culture Collection, and were cultured and differentiated as described previously ( ).

    Techniques: Transfection, Labeling, Fluorescence, Software, Confocal Microscopy

    Time course and dose course studies for the effect of exendin-4 on adiponectin expression in 3T3-L1 adipocytes. (A) Effects of exendin-4 on adiponectin mRNA expression at different time points (6, 12, 24, and 48 hours) and doses (0, 1.25, 2.5, and 5 nM). (B) Effects of exendin-4 on adiponectin protein expression at different time points and doses. (C) Effects of exendin-4 on the level of secreted adiponectin at different time points and doses. Statistical analysis shows that treating the cells with 2.5 nM exendin-4 for 24 hours provided the highest adiponectin level.

    Journal: PLoS ONE

    Article Title: Exendin-4 Upregulates Adiponectin Level in Adipocytes via Sirt1/Foxo-1 Signaling Pathway

    doi: 10.1371/journal.pone.0169469

    Figure Lengend Snippet: Time course and dose course studies for the effect of exendin-4 on adiponectin expression in 3T3-L1 adipocytes. (A) Effects of exendin-4 on adiponectin mRNA expression at different time points (6, 12, 24, and 48 hours) and doses (0, 1.25, 2.5, and 5 nM). (B) Effects of exendin-4 on adiponectin protein expression at different time points and doses. (C) Effects of exendin-4 on the level of secreted adiponectin at different time points and doses. Statistical analysis shows that treating the cells with 2.5 nM exendin-4 for 24 hours provided the highest adiponectin level.

    Article Snippet: Transfection of 3T3-L1 adipocytes 3T3-L1 adipocytes (ATCC, Cat. No. CL-173) were transfected with siRNA against GLP-R (Santa Cruz, Cat. No. sc-35382), Foxo-1 (Santa Cruz, Cat. No. 45760), Sirt1 (Santa Cruz sc-40986or their scramble controls using lipo RNAiMAX reagent (GIBCO, Cat. No 13778–150).

    Techniques: Expressing

    Gating strategies for cell sorting of CFDA-SE positive macrophage cells and effects of in vitro culture and collagenase treatment on C2D macrophage phenotype A) C2D macrophage cells were sorted based on negative expression of CFDA-SE, B) C2D CFDA-SE macrophage cells were sorted based on positive CFDA-SE expression; C) Example of C2D CFDA-SE + cells that were sorted from a mixed cell sample such as C2D CFDA-SE + macrophages (region 2) co-cultured with 3T3L1 adipocytes (region 1); D) PEC-C2D macrophages were treated with isotype control antibody (top) or anti Mac-2 antibody (middle and bottom) then assessed by flow cytometry. Cells were treated with PBS (middle) or collagenase (bottom) for 40 minutes before antibody probing.

    Journal: Cellular immunology

    Article Title: Evaluation of macrophage plasticity in brown and white adipose tissue

    doi: 10.1016/j.cellimm.2011.06.012

    Figure Lengend Snippet: Gating strategies for cell sorting of CFDA-SE positive macrophage cells and effects of in vitro culture and collagenase treatment on C2D macrophage phenotype A) C2D macrophage cells were sorted based on negative expression of CFDA-SE, B) C2D CFDA-SE macrophage cells were sorted based on positive CFDA-SE expression; C) Example of C2D CFDA-SE + cells that were sorted from a mixed cell sample such as C2D CFDA-SE + macrophages (region 2) co-cultured with 3T3L1 adipocytes (region 1); D) PEC-C2D macrophages were treated with isotype control antibody (top) or anti Mac-2 antibody (middle and bottom) then assessed by flow cytometry. Cells were treated with PBS (middle) or collagenase (bottom) for 40 minutes before antibody probing.

    Article Snippet: 3T3L1 adipocytes were obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: FACS, In Vitro, Expressing, Cell Culture, Flow Cytometry, Cytometry

    Change in C2D macrophage cell morphology during co-cultured with adipocytes or pre-adipocytes in vitro and after infiltration into BAT or WAT in vivo A) C2D macrophage cells were labeled with CFDA-SE or B) C2D macrophage cells labeled with CFDA-SE and isolated from peritoneal cavity (PEC-C2D) were cultured a) alone or co-cultured with b) 3T3L1 pre-adipocytes or c) adipocytes as described in the Materials and Methods. Panels a, b and c; Cells viewed on the fluorescent microscope (Magnification × 200). Panels d, e and f are phase contrast images of cells in a, b and c. C) WAT-C2D and BAT-C2D were collected from mice two days after adoptive transfer. C2 D macrophages, WAT and BAT were processed as described in Materials and Methods. Panels a and c images from the confocal microscope (× 100). Panels b and d are phase contrast images of the same fields.

    Journal: Cellular immunology

    Article Title: Evaluation of macrophage plasticity in brown and white adipose tissue

    doi: 10.1016/j.cellimm.2011.06.012

    Figure Lengend Snippet: Change in C2D macrophage cell morphology during co-cultured with adipocytes or pre-adipocytes in vitro and after infiltration into BAT or WAT in vivo A) C2D macrophage cells were labeled with CFDA-SE or B) C2D macrophage cells labeled with CFDA-SE and isolated from peritoneal cavity (PEC-C2D) were cultured a) alone or co-cultured with b) 3T3L1 pre-adipocytes or c) adipocytes as described in the Materials and Methods. Panels a, b and c; Cells viewed on the fluorescent microscope (Magnification × 200). Panels d, e and f are phase contrast images of cells in a, b and c. C) WAT-C2D and BAT-C2D were collected from mice two days after adoptive transfer. C2 D macrophages, WAT and BAT were processed as described in Materials and Methods. Panels a and c images from the confocal microscope (× 100). Panels b and d are phase contrast images of the same fields.

    Article Snippet: 3T3L1 adipocytes were obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Cell Culture, In Vitro, In Vivo, Labeling, Isolation, Microscopy, Mouse Assay, Adoptive Transfer Assay

    Phenotype changes of C2D macrophage cells co-cultured with adipocytes or pre-adipocytes in vitro C2D or PEC-C2D cells labeled with CFDA-SE were cultured alone or co-cultured with 3T3L1 adipocytes or pre-adipocytes andthe cell mixtures were immunostained for flow cytometry as described in Materials and Methods. C2D macrophage cells phenotypes were analyzed within CFDA-SE + population. A) C2D macrophage cells grown in vitro were cultured alone, co-cultured with 3T3L1 adipocytes or with pre-adipocytes. B) PEC-C2D macrophage cells were cultured alone, co-cultured with adipocytes or with pre-adipocytes. The data is presented as the mean ± SEM (n= 3 independently collected samples per treatment group). Different letters indicate a significant difference between control, preadipocytes or adipocytes for CD11b (lower case) or Mac-2 (upper case) cell surface proteins. A P value of

    Journal: Cellular immunology

    Article Title: Evaluation of macrophage plasticity in brown and white adipose tissue

    doi: 10.1016/j.cellimm.2011.06.012

    Figure Lengend Snippet: Phenotype changes of C2D macrophage cells co-cultured with adipocytes or pre-adipocytes in vitro C2D or PEC-C2D cells labeled with CFDA-SE were cultured alone or co-cultured with 3T3L1 adipocytes or pre-adipocytes andthe cell mixtures were immunostained for flow cytometry as described in Materials and Methods. C2D macrophage cells phenotypes were analyzed within CFDA-SE + population. A) C2D macrophage cells grown in vitro were cultured alone, co-cultured with 3T3L1 adipocytes or with pre-adipocytes. B) PEC-C2D macrophage cells were cultured alone, co-cultured with adipocytes or with pre-adipocytes. The data is presented as the mean ± SEM (n= 3 independently collected samples per treatment group). Different letters indicate a significant difference between control, preadipocytes or adipocytes for CD11b (lower case) or Mac-2 (upper case) cell surface proteins. A P value of

    Article Snippet: 3T3L1 adipocytes were obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Cell Culture, In Vitro, Labeling, Flow Cytometry, Cytometry

    Effects of ROS inhibitors on FFA-induced ER stress in 3T3-L1 adipocytes. A, Treatment with FFA up-regulated sXbp-1 mRNA in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 50 μg/ml FFA/BSA for 4 hours. uXbp-1 , unspliced forms of Xbp-1 mRNA; sXbp-1 , spliced forms of Xbp-1 mRNA. B, mRNA expression levels of ER stress markers in 3T3-L1 adipocytes treated with FFA. 3T3-L1 adipocytes were treated with 50 μg/ml FFA/BSA for 4 hours. mRNA expression levels ER stress markers were significantly increased in treatment with FFA. Values are mean ± SD (n = 3). *p

    Journal: Scientific Reports

    Article Title: Obesity-induced endoplasmic reticulum stress causes chronic inflammation in adipose tissue

    doi: 10.1038/srep00799

    Figure Lengend Snippet: Effects of ROS inhibitors on FFA-induced ER stress in 3T3-L1 adipocytes. A, Treatment with FFA up-regulated sXbp-1 mRNA in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 50 μg/ml FFA/BSA for 4 hours. uXbp-1 , unspliced forms of Xbp-1 mRNA; sXbp-1 , spliced forms of Xbp-1 mRNA. B, mRNA expression levels of ER stress markers in 3T3-L1 adipocytes treated with FFA. 3T3-L1 adipocytes were treated with 50 μg/ml FFA/BSA for 4 hours. mRNA expression levels ER stress markers were significantly increased in treatment with FFA. Values are mean ± SD (n = 3). *p

    Article Snippet: For experiments with chemical chaperones, 3T3-L1 adipocytes were treated with 7.5 mM 4-PBA or 0.5 mg/ml TUDCA for 14 h, followed by treatment with 0.6 μg/ml tunicamycin for 6 or 12 h. For ROS inhibitor studies, 3T3-L1 adipocytes were pretreated with 1 mM N -acetylcysteine (NAC) or 20 μM ebselen for 30 min, and then stimulated with 50 μg/ml FFA in the presence of 5% BSA (Sigma) for 4 h.

    Techniques: Expressing

    Effects of chemical chaperones on ER stress and the expression of inflammatory cytokines in 3T3-L1 adipocytes. A, Pretreatment with the chemical chaperones 4-PBA or TUDCA inhibited tunicamycin-induced up-regulation of sXbp-1 mRNA in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 7.5 mM 4-PBA or 0.5 mg/ml TUDCA for 14 hours, and then treated with 0.6 µg/ml tunicamycin for 6 hours. uXbp-1 , unspliced forms of Xbp-1 mRNA; sXbp-1 , spliced forms of Xbp-1 mRNA. B and C, Effects of 4-PBA or TUDCA on the mRNA expression of the ER stress markers Bip (B) and Chop (C) in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 7.5 mM 4-PBA or 0.5 mg/ml TUDCA for 14 hours, and then treated with 0.6 μg/ml tunicamycin for 12 hours. Pretreatment with 4-PBA and TUDCA prevented the up-regulation of Bip and Chop induced by tunicamycin. Values are mean ± SD (n = 3). *p

    Journal: Scientific Reports

    Article Title: Obesity-induced endoplasmic reticulum stress causes chronic inflammation in adipose tissue

    doi: 10.1038/srep00799

    Figure Lengend Snippet: Effects of chemical chaperones on ER stress and the expression of inflammatory cytokines in 3T3-L1 adipocytes. A, Pretreatment with the chemical chaperones 4-PBA or TUDCA inhibited tunicamycin-induced up-regulation of sXbp-1 mRNA in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 7.5 mM 4-PBA or 0.5 mg/ml TUDCA for 14 hours, and then treated with 0.6 µg/ml tunicamycin for 6 hours. uXbp-1 , unspliced forms of Xbp-1 mRNA; sXbp-1 , spliced forms of Xbp-1 mRNA. B and C, Effects of 4-PBA or TUDCA on the mRNA expression of the ER stress markers Bip (B) and Chop (C) in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 7.5 mM 4-PBA or 0.5 mg/ml TUDCA for 14 hours, and then treated with 0.6 μg/ml tunicamycin for 12 hours. Pretreatment with 4-PBA and TUDCA prevented the up-regulation of Bip and Chop induced by tunicamycin. Values are mean ± SD (n = 3). *p

    Article Snippet: For experiments with chemical chaperones, 3T3-L1 adipocytes were treated with 7.5 mM 4-PBA or 0.5 mg/ml TUDCA for 14 h, followed by treatment with 0.6 μg/ml tunicamycin for 6 or 12 h. For ROS inhibitor studies, 3T3-L1 adipocytes were pretreated with 1 mM N -acetylcysteine (NAC) or 20 μM ebselen for 30 min, and then stimulated with 50 μg/ml FFA in the presence of 5% BSA (Sigma) for 4 h.

    Techniques: Expressing

    Effects of DNMT1 knockdown in 3T3-L1 pre-adipocytes. A, B: DNMT1 mRNA (A) and protein (B). C, D: Promoter methylation (C) and leptin expression (D). E: Expression of the adipogenic genes, PPARγ, C/EBPα, aP2, GLUT4, adiponectin, and leptin. F: Cell proliferation. Results are mean ± SEM. *, p

    Journal: PLoS ONE

    Article Title: DNA Methylation Suppresses Leptin Gene in 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0160532

    Figure Lengend Snippet: Effects of DNMT1 knockdown in 3T3-L1 pre-adipocytes. A, B: DNMT1 mRNA (A) and protein (B). C, D: Promoter methylation (C) and leptin expression (D). E: Expression of the adipogenic genes, PPARγ, C/EBPα, aP2, GLUT4, adiponectin, and leptin. F: Cell proliferation. Results are mean ± SEM. *, p

    Article Snippet: To inhibit methylation, 3T3-L1 pre-adipocytes were cultured for seven days in DMEM supplemented with 10% calf serum and 0.5–5 μM 5-azacytidine (Sigma, MO, USA).

    Techniques: Methylation, Expressing

    Effects of adipocyte enlargement. A: CpG methylation in eWAT, liver, skeletal muscle, and brain tissue from mice 12 weeks of age. B: CpG methylation in adipocytes and stromal vascular fraction of whole adipose tissue. C: Methylation in adipocytes isolated from eWAT of mice on normal chow (NC) or high-fat (HF) diet. D: Time course of leptin expression in adipocytes differentiated from untreated or 5-azacytidine-treated 3T3-L1 precursors. E, F: Leptin promoter methylation at day 6 and 20 in adipocytes derived from untreated (E) and 5-azacytidine-treated (F) 3T3- L1 pre-adipocytes. Data are mean ± SEM. *, p

    Journal: PLoS ONE

    Article Title: DNA Methylation Suppresses Leptin Gene in 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0160532

    Figure Lengend Snippet: Effects of adipocyte enlargement. A: CpG methylation in eWAT, liver, skeletal muscle, and brain tissue from mice 12 weeks of age. B: CpG methylation in adipocytes and stromal vascular fraction of whole adipose tissue. C: Methylation in adipocytes isolated from eWAT of mice on normal chow (NC) or high-fat (HF) diet. D: Time course of leptin expression in adipocytes differentiated from untreated or 5-azacytidine-treated 3T3-L1 precursors. E, F: Leptin promoter methylation at day 6 and 20 in adipocytes derived from untreated (E) and 5-azacytidine-treated (F) 3T3- L1 pre-adipocytes. Data are mean ± SEM. *, p

    Article Snippet: To inhibit methylation, 3T3-L1 pre-adipocytes were cultured for seven days in DMEM supplemented with 10% calf serum and 0.5–5 μM 5-azacytidine (Sigma, MO, USA).

    Techniques: CpG Methylation Assay, Mouse Assay, Methylation, Isolation, Expressing, Derivative Assay

    Effects of demethylation on leptin expression in 3T3-L1 cells. A, B: Promoter methylation (A) and leptin expression (B) in 3T3-L1 pre-adipocytes treated with 0.5 or 5 μM 5-azacytidine for 7 days. C, D: Promoter methylation (C) and leptin expression (D) in adipocytes derived from 3T3-L1 precursors treated with or without 5-azacytidine, measured 6 days after differentiation. Products from real-time qPCR were also separated on agarose. E, F: Expression (E) and secretion (F) of leptin in adipocytes derived from precursors treated with 5-azacytidine, as measured by western blotting and ELISA, respectively. Data were normalized to total protein concentration. G: Activity of methylated and unmethylated leptin promoter in adipocytes differentiated from 3T3-L1 cells. H: Leptin expression in serum-starved cells treated with 100 nM insulin (Ins), 1 μM troglitazone (Tro), or 1 μM dexamethasone (Dex). Cells are adipocytes derived from 3T3-L1 precursors treated with 5-azacytidine. Results are mean ± SEM. *, p

    Journal: PLoS ONE

    Article Title: DNA Methylation Suppresses Leptin Gene in 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0160532

    Figure Lengend Snippet: Effects of demethylation on leptin expression in 3T3-L1 cells. A, B: Promoter methylation (A) and leptin expression (B) in 3T3-L1 pre-adipocytes treated with 0.5 or 5 μM 5-azacytidine for 7 days. C, D: Promoter methylation (C) and leptin expression (D) in adipocytes derived from 3T3-L1 precursors treated with or without 5-azacytidine, measured 6 days after differentiation. Products from real-time qPCR were also separated on agarose. E, F: Expression (E) and secretion (F) of leptin in adipocytes derived from precursors treated with 5-azacytidine, as measured by western blotting and ELISA, respectively. Data were normalized to total protein concentration. G: Activity of methylated and unmethylated leptin promoter in adipocytes differentiated from 3T3-L1 cells. H: Leptin expression in serum-starved cells treated with 100 nM insulin (Ins), 1 μM troglitazone (Tro), or 1 μM dexamethasone (Dex). Cells are adipocytes derived from 3T3-L1 precursors treated with 5-azacytidine. Results are mean ± SEM. *, p

    Article Snippet: To inhibit methylation, 3T3-L1 pre-adipocytes were cultured for seven days in DMEM supplemented with 10% calf serum and 0.5–5 μM 5-azacytidine (Sigma, MO, USA).

    Techniques: Expressing, Methylation, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration, Activity Assay

    Inhibitory effect of IBC on expression of adipogenic transcription factors. 3T3-L1 adipocytes were differentiated with IBC at indicated concentrations. On D5, cells were harvested and lysed to perform Western blotting analysis ( A ) and quantitative real time PCR (qPCR) ( B ). Data are presented as means ± SD of triplicate experiments. # p

    Journal: International Journal of Molecular Sciences

    Article Title: Isobavachalcone from Angelica keiskei Inhibits Adipogenesis and Prevents Lipid Accumulation

    doi: 10.3390/ijms19061693

    Figure Lengend Snippet: Inhibitory effect of IBC on expression of adipogenic transcription factors. 3T3-L1 adipocytes were differentiated with IBC at indicated concentrations. On D5, cells were harvested and lysed to perform Western blotting analysis ( A ) and quantitative real time PCR (qPCR) ( B ). Data are presented as means ± SD of triplicate experiments. # p

    Article Snippet: To measure cell cycle progression, confluent 3T3-L1 adipocytes were incubated with MDI in the presence or absence of IBC (40 μM) for 48 h and subjected to flow cytometry after staining with propidium iodide (PI) (Sigma).

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

    Effect of IBC on levels of lipid metabolism related genes. 3T3-L1 adipocytes were differentiated with IBC at indicated concentrations. On D5, cells were harvested and cell lysates were used for RNA extraction followed by cDNA synthesis and qPCR to analyze gene expression levels of SREBP1c ( A ); adiponectin ( B ); ACC1 ( C ); and FAS ( D ). Data are presented as means ± SD of triplicate experiments. # p

    Journal: International Journal of Molecular Sciences

    Article Title: Isobavachalcone from Angelica keiskei Inhibits Adipogenesis and Prevents Lipid Accumulation

    doi: 10.3390/ijms19061693

    Figure Lengend Snippet: Effect of IBC on levels of lipid metabolism related genes. 3T3-L1 adipocytes were differentiated with IBC at indicated concentrations. On D5, cells were harvested and cell lysates were used for RNA extraction followed by cDNA synthesis and qPCR to analyze gene expression levels of SREBP1c ( A ); adiponectin ( B ); ACC1 ( C ); and FAS ( D ). Data are presented as means ± SD of triplicate experiments. # p

    Article Snippet: To measure cell cycle progression, confluent 3T3-L1 adipocytes were incubated with MDI in the presence or absence of IBC (40 μM) for 48 h and subjected to flow cytometry after staining with propidium iodide (PI) (Sigma).

    Techniques: RNA Extraction, Real-time Polymerase Chain Reaction, Expressing

    Effect of IBC on autophagic flux during adipocyte differentiation. ( A ) 3T3-L1 adipocytes were differentiated in the presence or absence of 40 μM of IBC. Differentiating adipocytes were harvested at the indicated period and lysed for Western blotting analysis to determine levels of LC3B and SQSTM1/p62; ( B ) Preadipocytes were transfected with GFP-LC3 plasmid and differentiated with MDI in the presence of IBC or CQ (10 μM) during D0–D2, followed by additional treatment with differentiation medium for 2 days (D4). Cells were fixed and stained with DAPI (nuclei, blue-colored). Intracellular GFP-LC3 puncta (green-colored) were visualized with a confocal laser microscope. Scale bar = 10 μm; ( C ) Differentiating adipocytes supplemented with IBC or CQ during D0–D2 were harvested and protein levels of PPARγ and SQSTM1/p62 were analyzed on D8. Scale bar = 100 μm; and ( D ) On D2, differentiating adipocytes supplemented with IBC or CQ were harvested and subjected to qPCR to analyze gene expression levels of BECN1 , Atg5 , and Atg7 . Data are presented as means ± SD of triplicate experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Isobavachalcone from Angelica keiskei Inhibits Adipogenesis and Prevents Lipid Accumulation

    doi: 10.3390/ijms19061693

    Figure Lengend Snippet: Effect of IBC on autophagic flux during adipocyte differentiation. ( A ) 3T3-L1 adipocytes were differentiated in the presence or absence of 40 μM of IBC. Differentiating adipocytes were harvested at the indicated period and lysed for Western blotting analysis to determine levels of LC3B and SQSTM1/p62; ( B ) Preadipocytes were transfected with GFP-LC3 plasmid and differentiated with MDI in the presence of IBC or CQ (10 μM) during D0–D2, followed by additional treatment with differentiation medium for 2 days (D4). Cells were fixed and stained with DAPI (nuclei, blue-colored). Intracellular GFP-LC3 puncta (green-colored) were visualized with a confocal laser microscope. Scale bar = 10 μm; ( C ) Differentiating adipocytes supplemented with IBC or CQ during D0–D2 were harvested and protein levels of PPARγ and SQSTM1/p62 were analyzed on D8. Scale bar = 100 μm; and ( D ) On D2, differentiating adipocytes supplemented with IBC or CQ were harvested and subjected to qPCR to analyze gene expression levels of BECN1 , Atg5 , and Atg7 . Data are presented as means ± SD of triplicate experiments. * p

    Article Snippet: To measure cell cycle progression, confluent 3T3-L1 adipocytes were incubated with MDI in the presence or absence of IBC (40 μM) for 48 h and subjected to flow cytometry after staining with propidium iodide (PI) (Sigma).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Staining, Microscopy, Real-time Polymerase Chain Reaction, Expressing

    Effect of GST-irisin on the autocrine function and glucose concentration in 3T3-L1 mature adipose cells. A: Effect of GST-irisin on the genes upstream of itself (PPARγ and FNDC5) in 3T3-L1 mature adipocytes. The mRNA expression was determined by qPCR. B: The mature adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM) for 2 (a), 4 (b), 6 (c), and 8 (d) days. The irisin concentrations in the culture media in 3T3-L1 mature adipocytes were determined by ELISA. C: The GST-irisin concentration was 0 (white circles), 50 (white diamond), 100 (white triangle), and 200 (white squares) nM. D: The adipocytes were cultured with GST-irisin for 2 (a), 4 (b), 6 (c), and 8 (d) days. Values are expressed as the mean ± SD of 10 independent experiments. * P

    Journal: PLoS ONE

    Article Title: Effects and Molecular Mechanism of GST-Irisin on Lipolysis and Autocrine Function in 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0147480

    Figure Lengend Snippet: Effect of GST-irisin on the autocrine function and glucose concentration in 3T3-L1 mature adipose cells. A: Effect of GST-irisin on the genes upstream of itself (PPARγ and FNDC5) in 3T3-L1 mature adipocytes. The mRNA expression was determined by qPCR. B: The mature adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM) for 2 (a), 4 (b), 6 (c), and 8 (d) days. The irisin concentrations in the culture media in 3T3-L1 mature adipocytes were determined by ELISA. C: The GST-irisin concentration was 0 (white circles), 50 (white diamond), 100 (white triangle), and 200 (white squares) nM. D: The adipocytes were cultured with GST-irisin for 2 (a), 4 (b), 6 (c), and 8 (d) days. Values are expressed as the mean ± SD of 10 independent experiments. * P

    Article Snippet: The 3T3-L1 mature adipocytes were washed once with PBS and fixed with 3.7% formaldehyde (Sigma—Aldrich, USA) at room temperature for 20 min and then incubated with the staining solution for 1 h. The cells were washed twice with water.

    Techniques: Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effect of GST-irisin on lipolysis in 3T3-L1 mature adipocytes. A: Effect of GST-irisin on the release of glycerol in 3T3-L1 mature adipocytes. The adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM) for 2 (a), 4 (b), 6 (c) and 8 (d) days. B: The mRNA expression of FABP4, ATGL, and HSL was determined by qPCR. C: The protein levels of FABP4, ATGL, FNDC5, and β-actin were determined by western blotting. The adipocytes were cultured with various GST-irisin concentrations, and the concentration was 0 (a), 50 (b), 100 (c), and 200 (d) nM. Values are expressed as the mean ± SD of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Effects and Molecular Mechanism of GST-Irisin on Lipolysis and Autocrine Function in 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0147480

    Figure Lengend Snippet: Effect of GST-irisin on lipolysis in 3T3-L1 mature adipocytes. A: Effect of GST-irisin on the release of glycerol in 3T3-L1 mature adipocytes. The adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM) for 2 (a), 4 (b), 6 (c) and 8 (d) days. B: The mRNA expression of FABP4, ATGL, and HSL was determined by qPCR. C: The protein levels of FABP4, ATGL, FNDC5, and β-actin were determined by western blotting. The adipocytes were cultured with various GST-irisin concentrations, and the concentration was 0 (a), 50 (b), 100 (c), and 200 (d) nM. Values are expressed as the mean ± SD of three independent experiments. * P

    Article Snippet: The 3T3-L1 mature adipocytes were washed once with PBS and fixed with 3.7% formaldehyde (Sigma—Aldrich, USA) at room temperature for 20 min and then incubated with the staining solution for 1 h. The cells were washed twice with water.

    Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay

    Fat droplets in control and treated groups. 3T3-L1 mature adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM) for 2 (a), 4 (b), 6 (c) and 8 (d) days. A: Oil Red O staining (200×). B: OD values of isopropanol extraction. Values are expressed as the mean ± SD of three independent experiments. ** P

    Journal: PLoS ONE

    Article Title: Effects and Molecular Mechanism of GST-Irisin on Lipolysis and Autocrine Function in 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0147480

    Figure Lengend Snippet: Fat droplets in control and treated groups. 3T3-L1 mature adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM) for 2 (a), 4 (b), 6 (c) and 8 (d) days. A: Oil Red O staining (200×). B: OD values of isopropanol extraction. Values are expressed as the mean ± SD of three independent experiments. ** P

    Article Snippet: The 3T3-L1 mature adipocytes were washed once with PBS and fixed with 3.7% formaldehyde (Sigma—Aldrich, USA) at room temperature for 20 min and then incubated with the staining solution for 1 h. The cells were washed twice with water.

    Techniques: Cell Culture, Staining

    A: SDS-PAGE analysis of pGEX-4T-1–irisin expression and purification. M: low molecular weight marker (kDa); IN: induced culture; UI: un-induced culture; GST-Irisin: the purified eluent. B: Western Blotting of GST-irisin. IN: induced culture; UI: un-induced culture. C: Effect of GST-irisin on 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated with various GST-irisin concentrations (0, 12.5, 25, 50, 100, and 200 nM), and the cell viability was assessed using the CCK-8 assay. D: qPCR of UCP1 of 3T3-L1-derived adipocytes. 3T3-L1 mature adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM). The expression of UCP1 mRNA was determined by qPCR. Values are expressed as the mean ± SD of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Effects and Molecular Mechanism of GST-Irisin on Lipolysis and Autocrine Function in 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0147480

    Figure Lengend Snippet: A: SDS-PAGE analysis of pGEX-4T-1–irisin expression and purification. M: low molecular weight marker (kDa); IN: induced culture; UI: un-induced culture; GST-Irisin: the purified eluent. B: Western Blotting of GST-irisin. IN: induced culture; UI: un-induced culture. C: Effect of GST-irisin on 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated with various GST-irisin concentrations (0, 12.5, 25, 50, 100, and 200 nM), and the cell viability was assessed using the CCK-8 assay. D: qPCR of UCP1 of 3T3-L1-derived adipocytes. 3T3-L1 mature adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM). The expression of UCP1 mRNA was determined by qPCR. Values are expressed as the mean ± SD of three independent experiments. * P

    Article Snippet: The 3T3-L1 mature adipocytes were washed once with PBS and fixed with 3.7% formaldehyde (Sigma—Aldrich, USA) at room temperature for 20 min and then incubated with the staining solution for 1 h. The cells were washed twice with water.

    Techniques: SDS Page, Expressing, Purification, Molecular Weight, Marker, Western Blot, CCK-8 Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Cell Culture

    Effect of ABG extract on glycerol release in mature 3T3-L1 adipocytes. Extracellular free glycerol was measured with a glycerol quantification kit. Values are expressed as mean ± SD of three separate experiments. ** p

    Journal: Food Science and Biotechnology

    Article Title: Aged black garlic extract regulates lipid metabolism by inhibiting lipogenesis and promoting lipolysis in mature 3T3-L1 adipocytes

    doi: 10.1007/s10068-017-0268-y

    Figure Lengend Snippet: Effect of ABG extract on glycerol release in mature 3T3-L1 adipocytes. Extracellular free glycerol was measured with a glycerol quantification kit. Values are expressed as mean ± SD of three separate experiments. ** p

    Article Snippet: Mature 3T3-L1 adipocytes were treated with ABG extract for 48 h. Released free glycerol was then assayed using the Glycerol Assay Kit according to the manufacturer’s protocol (MAK117, Sigma-Aldrich, St. Louis, MO, USA).

    Techniques:

    Effect of ABG extract on the expression of lipogenic and lipolytic proteins in mature 3T3-L1 adipocytes. Expression levels of ( A ) PPARγ, ( B ) p-HSL-Ser 563 and HSL, and ( C ) perilipin are shown. Band intensities of PPARγ, HSL, and perilipin were normalized to their respective ß-actin fractions, whereas p-HSL-Ser 563 was normalized to HSL levels. Values are expressed as mean ± SD of three separate experiments. ** p

    Journal: Food Science and Biotechnology

    Article Title: Aged black garlic extract regulates lipid metabolism by inhibiting lipogenesis and promoting lipolysis in mature 3T3-L1 adipocytes

    doi: 10.1007/s10068-017-0268-y

    Figure Lengend Snippet: Effect of ABG extract on the expression of lipogenic and lipolytic proteins in mature 3T3-L1 adipocytes. Expression levels of ( A ) PPARγ, ( B ) p-HSL-Ser 563 and HSL, and ( C ) perilipin are shown. Band intensities of PPARγ, HSL, and perilipin were normalized to their respective ß-actin fractions, whereas p-HSL-Ser 563 was normalized to HSL levels. Values are expressed as mean ± SD of three separate experiments. ** p

    Article Snippet: Mature 3T3-L1 adipocytes were treated with ABG extract for 48 h. Released free glycerol was then assayed using the Glycerol Assay Kit according to the manufacturer’s protocol (MAK117, Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Expressing

    Effects of ABG extract on viability and lipid accumulation in mature 3T3-L1 adipocytes. (A) Mature 3T3-L1 adipocytes were incubated for 72 h with various concentrations of ABG extract (0–5.0 mg/mL). Then cell viability assay was performed using CCK-8 assay kit. ( B ) Mature 3T3-L1 adipocytes were stained with Oil Red O after 7 days of treatment with 0-5.0 mg/mL of ABG extract. ( C ) Microscopic pictures were taken at 200 × magnification. The OD values were measured from isopropanol elution of the Oil Red O stained cells. ** p

    Journal: Food Science and Biotechnology

    Article Title: Aged black garlic extract regulates lipid metabolism by inhibiting lipogenesis and promoting lipolysis in mature 3T3-L1 adipocytes

    doi: 10.1007/s10068-017-0268-y

    Figure Lengend Snippet: Effects of ABG extract on viability and lipid accumulation in mature 3T3-L1 adipocytes. (A) Mature 3T3-L1 adipocytes were incubated for 72 h with various concentrations of ABG extract (0–5.0 mg/mL). Then cell viability assay was performed using CCK-8 assay kit. ( B ) Mature 3T3-L1 adipocytes were stained with Oil Red O after 7 days of treatment with 0-5.0 mg/mL of ABG extract. ( C ) Microscopic pictures were taken at 200 × magnification. The OD values were measured from isopropanol elution of the Oil Red O stained cells. ** p

    Article Snippet: Mature 3T3-L1 adipocytes were treated with ABG extract for 48 h. Released free glycerol was then assayed using the Glycerol Assay Kit according to the manufacturer’s protocol (MAK117, Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Incubation, Viability Assay, CCK-8 Assay, Staining

    Munc18c residues Tyr219 and Tyr521 are essential for insulin-stimulated GLUT4 vesicle exocytosis. (A) 3T3-L1 adipocytes coelectroporated with Munc18c-specific (siMunc18c) or nontargeting control (siCon) shRNA plus Myc-GLUT4-GFP plasmid DNAs were left unstimulated or were stimulated with 100 nM insulin for 30 min followed by fixation but not permeabilization. Exofacial Myc exposure was determined by anti-Myc immunostaining (red) followed by immunofluorescent confocal microscopy. GFP fluorescence was examined to indicate subcellular location of GLUT4-GFP vesicles (green). The number of Myc-stained cells relative to the total number of GFP-fluorescing cells (≥50 cells per condition) was quantified in at least three independent sets of cells as depicted in the bar graph inset; *, P

    Journal: The Journal of Cell Biology

    Article Title: Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

    doi: 10.1083/jcb.201007176

    Figure Lengend Snippet: Munc18c residues Tyr219 and Tyr521 are essential for insulin-stimulated GLUT4 vesicle exocytosis. (A) 3T3-L1 adipocytes coelectroporated with Munc18c-specific (siMunc18c) or nontargeting control (siCon) shRNA plus Myc-GLUT4-GFP plasmid DNAs were left unstimulated or were stimulated with 100 nM insulin for 30 min followed by fixation but not permeabilization. Exofacial Myc exposure was determined by anti-Myc immunostaining (red) followed by immunofluorescent confocal microscopy. GFP fluorescence was examined to indicate subcellular location of GLUT4-GFP vesicles (green). The number of Myc-stained cells relative to the total number of GFP-fluorescing cells (≥50 cells per condition) was quantified in at least three independent sets of cells as depicted in the bar graph inset; *, P

    Article Snippet: Unlike the islet β cell, which expresses and utilizes multiple Munc18–syntaxin complexes for multiple phases of insulin release, 3T3-L1 adipocytes use only the Munc18c–syntaxin 4 pair for the monophasic process of insulin-stimulated GLUT4 vesicle exocytosis ( ; ; ; ).

    Techniques: shRNA, Plasmid Preparation, Immunostaining, Confocal Microscopy, Fluorescence, Staining

    Insulin stimulates the association of IR with Munc18c. (A) Munc18c was immunoprecipitated (IP) from cleared detergent lysates prepared from fully differentiated 3T3-L1 adipocytes that were incubated in serum-free medium for 2 h and then stimulated with 100 nM insulin for 5 min. Proteins were resolved by 10% SDS-PAGE and immunoblotted (IB) with anti-Munc18c and anti–insulin receptor (IR) antibodies. Data are from three independent sets of lysates quantified as the ratio of IR–Munc18c, with each set normalized to basal = 1.0; *, P

    Journal: The Journal of Cell Biology

    Article Title: Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

    doi: 10.1083/jcb.201007176

    Figure Lengend Snippet: Insulin stimulates the association of IR with Munc18c. (A) Munc18c was immunoprecipitated (IP) from cleared detergent lysates prepared from fully differentiated 3T3-L1 adipocytes that were incubated in serum-free medium for 2 h and then stimulated with 100 nM insulin for 5 min. Proteins were resolved by 10% SDS-PAGE and immunoblotted (IB) with anti-Munc18c and anti–insulin receptor (IR) antibodies. Data are from three independent sets of lysates quantified as the ratio of IR–Munc18c, with each set normalized to basal = 1.0; *, P

    Article Snippet: Unlike the islet β cell, which expresses and utilizes multiple Munc18–syntaxin complexes for multiple phases of insulin release, 3T3-L1 adipocytes use only the Munc18c–syntaxin 4 pair for the monophasic process of insulin-stimulated GLUT4 vesicle exocytosis ( ; ; ; ).

    Techniques: Immunoprecipitation, Incubation, SDS Page

    Tyrosine phosphorylation of Munc18c maps to a motif centered at Tyr219 in adipocytes. Fully differentiated 3T3-L1 adipocytes were preincubated in serum-free media for 2 h followed by treatment with freshly prepared 0.1 mM pervanadate (pV) for 5 min. (A) Cells were immediately fixed and permeabilized for immunostaining with a phosphospecific anti–p Y219 -Munc18c peptide antibody in the absence of a competitive peptide (image 1). The specificity of the antibody for the Y219-centered phosphopeptide was assessed by competition with the addition of 500 ng of the antigenic peptide in its nonphosphorylated (Non–P-Tyr, image 2) or phosphorylated (P-Tyr, image 3) state. Image 4 validates the presence of cells in the field imaged in image 3. Immunofluorescent confocal microscopy was used to visualize ≥50 cells each. Bars, 10 µm. (B) Cleared detergent lysates were prepared for anti-Munc18c immunoprecipitation (IP). Protein was resolved on 10% SDS-PAGE for immunoblotting (IB) with anti–p Y219 -Munc18c antibody. Ponceau S staining shows equal loading of the immunoprecipitation reactions. All data are representative of two to three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

    doi: 10.1083/jcb.201007176

    Figure Lengend Snippet: Tyrosine phosphorylation of Munc18c maps to a motif centered at Tyr219 in adipocytes. Fully differentiated 3T3-L1 adipocytes were preincubated in serum-free media for 2 h followed by treatment with freshly prepared 0.1 mM pervanadate (pV) for 5 min. (A) Cells were immediately fixed and permeabilized for immunostaining with a phosphospecific anti–p Y219 -Munc18c peptide antibody in the absence of a competitive peptide (image 1). The specificity of the antibody for the Y219-centered phosphopeptide was assessed by competition with the addition of 500 ng of the antigenic peptide in its nonphosphorylated (Non–P-Tyr, image 2) or phosphorylated (P-Tyr, image 3) state. Image 4 validates the presence of cells in the field imaged in image 3. Immunofluorescent confocal microscopy was used to visualize ≥50 cells each. Bars, 10 µm. (B) Cleared detergent lysates were prepared for anti-Munc18c immunoprecipitation (IP). Protein was resolved on 10% SDS-PAGE for immunoblotting (IB) with anti–p Y219 -Munc18c antibody. Ponceau S staining shows equal loading of the immunoprecipitation reactions. All data are representative of two to three independent experiments.

    Article Snippet: Unlike the islet β cell, which expresses and utilizes multiple Munc18–syntaxin complexes for multiple phases of insulin release, 3T3-L1 adipocytes use only the Munc18c–syntaxin 4 pair for the monophasic process of insulin-stimulated GLUT4 vesicle exocytosis ( ; ; ; ).

    Techniques: Immunostaining, Confocal Microscopy, Immunoprecipitation, SDS Page, Staining

    Insulin stimulates the tyrosine phosphorylation of Munc18c in a PI3K-independent pathway. Fully differentiated 3T3-L1 adipocytes were preincubated in serum-free media for 2 h with or without 100 nM wortmannin followed by 5-min insulin stimulation (100 nM) and subsequent preparation of cleared detergent cell lysates. Munc18c was immunoprecipitated (IP) from lysates, protein was resolved by 10% SDS-PAGE, and tyrosine-phosphorylated (P-Tyr) and total Munc18c was detected by immunoblotting (IB) with anti-4G10 or PY20 antibodies and anti-Munc18c, respectively; this experiment was performed by reprobing of the same blot with both antibodies. Quantitation of tyrosine-phosphorylated Munc18c/total Munc18c from at least three independent cell passages, with each experiment normalized to insulin stimulation minus wortmannin = 1.0. Insulin and wortmannin actions in lysates used for immunoprecipitation were validated by detection of phosphorylated Akt (P-Akt) content, relative to total Akt. Data represent means ± SEM. Black lines indicate that intervening lanes have been spliced out.

    Journal: The Journal of Cell Biology

    Article Title: Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

    doi: 10.1083/jcb.201007176

    Figure Lengend Snippet: Insulin stimulates the tyrosine phosphorylation of Munc18c in a PI3K-independent pathway. Fully differentiated 3T3-L1 adipocytes were preincubated in serum-free media for 2 h with or without 100 nM wortmannin followed by 5-min insulin stimulation (100 nM) and subsequent preparation of cleared detergent cell lysates. Munc18c was immunoprecipitated (IP) from lysates, protein was resolved by 10% SDS-PAGE, and tyrosine-phosphorylated (P-Tyr) and total Munc18c was detected by immunoblotting (IB) with anti-4G10 or PY20 antibodies and anti-Munc18c, respectively; this experiment was performed by reprobing of the same blot with both antibodies. Quantitation of tyrosine-phosphorylated Munc18c/total Munc18c from at least three independent cell passages, with each experiment normalized to insulin stimulation minus wortmannin = 1.0. Insulin and wortmannin actions in lysates used for immunoprecipitation were validated by detection of phosphorylated Akt (P-Akt) content, relative to total Akt. Data represent means ± SEM. Black lines indicate that intervening lanes have been spliced out.

    Article Snippet: Unlike the islet β cell, which expresses and utilizes multiple Munc18–syntaxin complexes for multiple phases of insulin release, 3T3-L1 adipocytes use only the Munc18c–syntaxin 4 pair for the monophasic process of insulin-stimulated GLUT4 vesicle exocytosis ( ; ; ; ).

    Techniques: Immunoprecipitation, SDS Page, Quantitation Assay

    pV induces IR–Munc18c complex formation and Munc18c–syntaxin 4 dissociation. (A–C) Cleared detergent lysates prepared from fully differentiated 3T3-L1 adipocytes that were incubated in serum-free medium for 2 h and then treated with or without 0.1 mM of freshly prepared pervanadate (pV) for 5 min were used in coimmunoprecipitation (IP) reactions: anti-Munc18c (A), anti-IR (B), or anti–syntaxin 4 (C, Syn4). Anti-Munc18c and anti–syntaxin 4 immunoprecipitation reactions were processed in parallel from the same starting lysates, which were confirmed to contain equivalent Munc18c protein (lysates). Munc18c and syntaxin 4 abundances in corresponding starting lysates used for B were also confirmed (Lysates). Coimmunoprecipitated proteins were resolved on 10% SDS-PAGE for immunoblotting (IB) with anti-Munc18c, anti-IR, and anti–syntaxin 4 antibodies; the Munc18c blot was stripped and reprobed with antiphosphotyrosine 4G10 (P-Tyr). Data represent the means ± SEM from three independent experiments for each type of immunoprecipitation, quantified as the ratio of IR/total Munc18c (A, i ), tyrosine-phosphorylated Munc18c/total Munc18c (A, ii ), or Munc18c/total syntaxin 4 (C) immunoprecipitated, with each set normalized to untreated = 1.0; *, P

    Journal: The Journal of Cell Biology

    Article Title: Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

    doi: 10.1083/jcb.201007176

    Figure Lengend Snippet: pV induces IR–Munc18c complex formation and Munc18c–syntaxin 4 dissociation. (A–C) Cleared detergent lysates prepared from fully differentiated 3T3-L1 adipocytes that were incubated in serum-free medium for 2 h and then treated with or without 0.1 mM of freshly prepared pervanadate (pV) for 5 min were used in coimmunoprecipitation (IP) reactions: anti-Munc18c (A), anti-IR (B), or anti–syntaxin 4 (C, Syn4). Anti-Munc18c and anti–syntaxin 4 immunoprecipitation reactions were processed in parallel from the same starting lysates, which were confirmed to contain equivalent Munc18c protein (lysates). Munc18c and syntaxin 4 abundances in corresponding starting lysates used for B were also confirmed (Lysates). Coimmunoprecipitated proteins were resolved on 10% SDS-PAGE for immunoblotting (IB) with anti-Munc18c, anti-IR, and anti–syntaxin 4 antibodies; the Munc18c blot was stripped and reprobed with antiphosphotyrosine 4G10 (P-Tyr). Data represent the means ± SEM from three independent experiments for each type of immunoprecipitation, quantified as the ratio of IR/total Munc18c (A, i ), tyrosine-phosphorylated Munc18c/total Munc18c (A, ii ), or Munc18c/total syntaxin 4 (C) immunoprecipitated, with each set normalized to untreated = 1.0; *, P

    Article Snippet: Unlike the islet β cell, which expresses and utilizes multiple Munc18–syntaxin complexes for multiple phases of insulin release, 3T3-L1 adipocytes use only the Munc18c–syntaxin 4 pair for the monophasic process of insulin-stimulated GLUT4 vesicle exocytosis ( ; ; ; ).

    Techniques: Incubation, Immunoprecipitation, SDS Page

    IR kinase activity is required to evoke IR binding and tyrosine phosphorylation of Munc18c. Fully differentiated 3T3-L1 adipocytes were preincubated in serum-free media for 1 h followed by an additional 1 h with or without the IR kinase activity inhibitor HNMPA-(AM) 3 (100 µM) before insulin stimulation for 5 min. (A) Cleared detergent lysates were prepared and used for Munc18c immunoprecipitation (IP) and detection of tyrosine-phosphorylated Munc18c (P-Tyr) using phosphotyrosine-specific 4G10 or PY20 antibodies; this experiment was performed by reprobing of the same blot with both antibodies. (B) HNMPA-(AM) 3 action was validated by detection of reduced Akt phosphorylation in the lysates used for immunoprecipitation; this experiment was performed by reprobing of the same blot with both antibodies. (C) IR is coimmunoprecipitated by anti-Munc18c. Coimmunoprecipitated proteins were resolved on 10% SDS-PAGE for immunoblotting (IB) with anti-Munc18c and anti-IR antibodies. IR abundance in corresponding starting lysates was confirmed by immunoblotting (Lysate). Data represent the means ± SEM from three independent sets of lysates for each panel, with each set normalized to basal = 1.0 (A) or insulin stimulation without HNMPA-(AM) 3 = 1.0 (C); *, P

    Journal: The Journal of Cell Biology

    Article Title: Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

    doi: 10.1083/jcb.201007176

    Figure Lengend Snippet: IR kinase activity is required to evoke IR binding and tyrosine phosphorylation of Munc18c. Fully differentiated 3T3-L1 adipocytes were preincubated in serum-free media for 1 h followed by an additional 1 h with or without the IR kinase activity inhibitor HNMPA-(AM) 3 (100 µM) before insulin stimulation for 5 min. (A) Cleared detergent lysates were prepared and used for Munc18c immunoprecipitation (IP) and detection of tyrosine-phosphorylated Munc18c (P-Tyr) using phosphotyrosine-specific 4G10 or PY20 antibodies; this experiment was performed by reprobing of the same blot with both antibodies. (B) HNMPA-(AM) 3 action was validated by detection of reduced Akt phosphorylation in the lysates used for immunoprecipitation; this experiment was performed by reprobing of the same blot with both antibodies. (C) IR is coimmunoprecipitated by anti-Munc18c. Coimmunoprecipitated proteins were resolved on 10% SDS-PAGE for immunoblotting (IB) with anti-Munc18c and anti-IR antibodies. IR abundance in corresponding starting lysates was confirmed by immunoblotting (Lysate). Data represent the means ± SEM from three independent sets of lysates for each panel, with each set normalized to basal = 1.0 (A) or insulin stimulation without HNMPA-(AM) 3 = 1.0 (C); *, P

    Article Snippet: Unlike the islet β cell, which expresses and utilizes multiple Munc18–syntaxin complexes for multiple phases of insulin release, 3T3-L1 adipocytes use only the Munc18c–syntaxin 4 pair for the monophasic process of insulin-stimulated GLUT4 vesicle exocytosis ( ; ; ; ).

    Techniques: Activity Assay, Binding Assay, Immunoprecipitation, SDS Page

    Insulin stimulation increases Munc18c tyrosine phosphorylation. (A) Munc18c was immunoprecipitated (IP) from cleared detergent cell lysates prepared from fully differentiated 3T3-L1 adipocytes that were incubated in serum-free medium for 2 h and then stimulated with 100 nM insulin for 5 min. Proteins were resolved by 10% SDS-PAGE, immunoblotted (IB) for Munc18c, stripped, and reblotted for detection of tyrosine-phosphorylated (P-Tyr) Munc18c using the 4G10 antibody. Data were collected from four independent experiments quantified as the ratio of tyrosine-phosphorylated Munc18c/total Munc18c precipitated and normalized to basal set = 1.0; *, P

    Journal: The Journal of Cell Biology

    Article Title: Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

    doi: 10.1083/jcb.201007176

    Figure Lengend Snippet: Insulin stimulation increases Munc18c tyrosine phosphorylation. (A) Munc18c was immunoprecipitated (IP) from cleared detergent cell lysates prepared from fully differentiated 3T3-L1 adipocytes that were incubated in serum-free medium for 2 h and then stimulated with 100 nM insulin for 5 min. Proteins were resolved by 10% SDS-PAGE, immunoblotted (IB) for Munc18c, stripped, and reblotted for detection of tyrosine-phosphorylated (P-Tyr) Munc18c using the 4G10 antibody. Data were collected from four independent experiments quantified as the ratio of tyrosine-phosphorylated Munc18c/total Munc18c precipitated and normalized to basal set = 1.0; *, P

    Article Snippet: Unlike the islet β cell, which expresses and utilizes multiple Munc18–syntaxin complexes for multiple phases of insulin release, 3T3-L1 adipocytes use only the Munc18c–syntaxin 4 pair for the monophasic process of insulin-stimulated GLUT4 vesicle exocytosis ( ; ; ; ).

    Techniques: Immunoprecipitation, Incubation, SDS Page

    Generation of a novel stable 3T3-L1 adipocyte model of insulin receptoropathy. ( a ) Concatenated miR-shRNAs targeting murine Insr in exon 2 and exon 9 preceded by GFP under the control of a tet-responsive element was packaged into third-generation lentivirus to enable transduction of 3T3-L1 pre-adipocytes. The exploded view shows the nucleotide mismatches between the mouse Insr targeted by each miR-shRNA with the human INSR sequence. Green shaded elements of the transgene are inducible by the addition of DOX. Transduced 3T3-L1 pre-adipocytes underwent single cell clonal selection in the presence of hygromycin to generate 3T3-L1 MmINSRKD. ( b ) 3T3-L1 MmINSRKD cells were then transduced with a second lentivirus encoding C-terminal myc-tagged human INSR transgenes under the control of a tet-responsive element and underwent polyclonal selection in the presence of neomycin to generate 3T3-L1 MmINSRKD hINSR . ( c ) Western blots of whole-cell lysates from day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT cells grown in the presence of increasing concentrations of DOX for 72 h. ( d ) Densitometry analysis of western blots from three independent experiments demonstrating knockdown of endogenous mouse Insr and expression of human INSR with increasing concentrations of DOX. ( e ) Western blots of whole-cell lysates from day 16 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR (mutant INSR as indicated) cells grown in the presence of 1 μg/ml DOX for 10 days. ( f ) Oil Red O staining of lipid accumulation in day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT or mutant (as indicated) cells grown ± DOX (1 μg/ml) for 72 h. GFP, green fluorescent protein; Hygro, hygromycin resistance; IRES, internal ribosome entry site; Mm, murine; Neo, neomycin resistance; rtTA3, reverse tetracycline-controlled transactivator; TRE, tet-response element; Ubi-C, ubiquitin C promoter

    Journal: Diabetologia

    Article Title: Evaluation of anti-insulin receptor antibodies as potential novel therapies for human insulin receptoropathy using cell culture models

    doi: 10.1007/s00125-018-4606-2

    Figure Lengend Snippet: Generation of a novel stable 3T3-L1 adipocyte model of insulin receptoropathy. ( a ) Concatenated miR-shRNAs targeting murine Insr in exon 2 and exon 9 preceded by GFP under the control of a tet-responsive element was packaged into third-generation lentivirus to enable transduction of 3T3-L1 pre-adipocytes. The exploded view shows the nucleotide mismatches between the mouse Insr targeted by each miR-shRNA with the human INSR sequence. Green shaded elements of the transgene are inducible by the addition of DOX. Transduced 3T3-L1 pre-adipocytes underwent single cell clonal selection in the presence of hygromycin to generate 3T3-L1 MmINSRKD. ( b ) 3T3-L1 MmINSRKD cells were then transduced with a second lentivirus encoding C-terminal myc-tagged human INSR transgenes under the control of a tet-responsive element and underwent polyclonal selection in the presence of neomycin to generate 3T3-L1 MmINSRKD hINSR . ( c ) Western blots of whole-cell lysates from day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT cells grown in the presence of increasing concentrations of DOX for 72 h. ( d ) Densitometry analysis of western blots from three independent experiments demonstrating knockdown of endogenous mouse Insr and expression of human INSR with increasing concentrations of DOX. ( e ) Western blots of whole-cell lysates from day 16 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR (mutant INSR as indicated) cells grown in the presence of 1 μg/ml DOX for 10 days. ( f ) Oil Red O staining of lipid accumulation in day 10 mature 3T3-L1 MmINSRKD and 3T3-L1 MmINSRKD hINSR WT or mutant (as indicated) cells grown ± DOX (1 μg/ml) for 72 h. GFP, green fluorescent protein; Hygro, hygromycin resistance; IRES, internal ribosome entry site; Mm, murine; Neo, neomycin resistance; rtTA3, reverse tetracycline-controlled transactivator; TRE, tet-response element; Ubi-C, ubiquitin C promoter

    Article Snippet: A tetracycline (tet)-responsive microRNA (miR)-short hairpin (sh)RNA selectively targeting murine Insr was transduced into 3T3-L1 pre-adipocytes to generate a stable clone (Fig. a).

    Techniques: Transduction, shRNA, Sequencing, Selection, Western Blot, Expressing, Mutagenesis, Staining

    PTN and PTHR role in mammary epithelial cell migration A) Mammary epithelial cells, HC-11 and differentiated 3T3-L1 adipocytes were lysed and tested for the presence of PTN, and its receptor PTPRζ1 by Western blotting. Left panel: PTN is highly expressed (upper panel) by 3T3-L1 adipocytes, it is undetectable in HC-11 cells; PTN-receptor PTPRζ1 is highly expressed in HC-11 cells but minimally expressed in the 3T3-L1 adipocytes (middle left panel). Both tubulin and vinculin serve as loading control because of difference in their expression between the cell types. Right panel: differentiated cell lysates (1/5 of total) and serum-free conditioned medium (1/100 of total), collected after 48hrs of incubation, tested for PTN by Western blotting. B) PTN was knocked down in differentiated 3T3-L1 adipocytes by pooled siRNA and C) PTHR was knocked down in 3T3-L1 using pooled PTHR-siRNA as described in Methods (middle panel). The treatments impaired cell migration of HC-11 cells in Boyden chambers (upper panels) (p

    Journal: Developmental biology

    Article Title: Adipocyte Derived Paracrine Mediators of Mammary Ductal Morphogenesis Controlled by Retinoic Acid Receptors

    doi: 10.1016/j.ydbio.2010.10.018

    Figure Lengend Snippet: PTN and PTHR role in mammary epithelial cell migration A) Mammary epithelial cells, HC-11 and differentiated 3T3-L1 adipocytes were lysed and tested for the presence of PTN, and its receptor PTPRζ1 by Western blotting. Left panel: PTN is highly expressed (upper panel) by 3T3-L1 adipocytes, it is undetectable in HC-11 cells; PTN-receptor PTPRζ1 is highly expressed in HC-11 cells but minimally expressed in the 3T3-L1 adipocytes (middle left panel). Both tubulin and vinculin serve as loading control because of difference in their expression between the cell types. Right panel: differentiated cell lysates (1/5 of total) and serum-free conditioned medium (1/100 of total), collected after 48hrs of incubation, tested for PTN by Western blotting. B) PTN was knocked down in differentiated 3T3-L1 adipocytes by pooled siRNA and C) PTHR was knocked down in 3T3-L1 using pooled PTHR-siRNA as described in Methods (middle panel). The treatments impaired cell migration of HC-11 cells in Boyden chambers (upper panels) (p

    Article Snippet: 3T3-L1 adipocytes promote the growth of mammary epithelium.

    Techniques: Migration, Western Blot, Expressing, Incubation

    RAR regulated genes in differentiated 3T3-L1 adipocytes and in mammary adipocytes in vivo A) 3T3-L1 cells were allowed to fully differentiate and were incubated in charcoal-stripped serum for 4 days. Cells were either treated for 48hr with 1μM atRA or left untreated and RNA was extracted. PTHR and PTN (and β2, as positive control) expression was detected using RT-PCR. GAPDH served as loading control. B) Differentiated 3T3-L1 cells were transfected either with empty vector or with DN-RARα plasmid and following overnight incubation, cell lysates were prepared and examined by Western blotting using anti-HA antibody (to detect transgene expression), or anti-PTHR antibody (left panel), or anti-PTN antibody (right panel). Tubulin served as loading control. Both PTHR and PTN levels were reduced in DN-RAR transfected adipocytes. C) Vector or DN-RARα-transfected, differentiated 3T3-L1 cells were treated with 200 and 500nM of atRA for 24hrs, the protein was extracted and tested for PTN expression. Tubulin served as loading control. D. Mammary glands #3 and #4 of wt-mice or Tg-mice untreated or treated daily with 4mg atRA for 72hrs, were removed, the RNA was extracted and analyzed for RARβ and PTN using Q-PCR. The bar represents an average of glands from 4 mice.

    Journal: Developmental biology

    Article Title: Adipocyte Derived Paracrine Mediators of Mammary Ductal Morphogenesis Controlled by Retinoic Acid Receptors

    doi: 10.1016/j.ydbio.2010.10.018

    Figure Lengend Snippet: RAR regulated genes in differentiated 3T3-L1 adipocytes and in mammary adipocytes in vivo A) 3T3-L1 cells were allowed to fully differentiate and were incubated in charcoal-stripped serum for 4 days. Cells were either treated for 48hr with 1μM atRA or left untreated and RNA was extracted. PTHR and PTN (and β2, as positive control) expression was detected using RT-PCR. GAPDH served as loading control. B) Differentiated 3T3-L1 cells were transfected either with empty vector or with DN-RARα plasmid and following overnight incubation, cell lysates were prepared and examined by Western blotting using anti-HA antibody (to detect transgene expression), or anti-PTHR antibody (left panel), or anti-PTN antibody (right panel). Tubulin served as loading control. Both PTHR and PTN levels were reduced in DN-RAR transfected adipocytes. C) Vector or DN-RARα-transfected, differentiated 3T3-L1 cells were treated with 200 and 500nM of atRA for 24hrs, the protein was extracted and tested for PTN expression. Tubulin served as loading control. D. Mammary glands #3 and #4 of wt-mice or Tg-mice untreated or treated daily with 4mg atRA for 72hrs, were removed, the RNA was extracted and analyzed for RARβ and PTN using Q-PCR. The bar represents an average of glands from 4 mice.

    Article Snippet: 3T3-L1 adipocytes promote the growth of mammary epithelium.

    Techniques: In Vivo, Incubation, Positive Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, Mouse Assay, Polymerase Chain Reaction

    The expression and localization of the exocyst complex in adipocytes. (A) The mRNA expression of Exo70, Sec6, and Sec8 before and after 3T3-L1 adipocyte differentiation. Pread: 3T1-L1 preadipocytes, Ad: 3T3-L1 adipocytes. (B) Upper, intracellular localization of mCherry-Exo70 (red) and lipid droplets stained with BODIPY 493/503 (green); lower, HA-Sec8 (red) and lipid droplets (green). Scale = 10 μm.

    Journal: PLoS ONE

    Article Title: The Exocyst Complex Regulates Free Fatty Acid Uptake by Adipocytes

    doi: 10.1371/journal.pone.0120289

    Figure Lengend Snippet: The expression and localization of the exocyst complex in adipocytes. (A) The mRNA expression of Exo70, Sec6, and Sec8 before and after 3T3-L1 adipocyte differentiation. Pread: 3T1-L1 preadipocytes, Ad: 3T3-L1 adipocytes. (B) Upper, intracellular localization of mCherry-Exo70 (red) and lipid droplets stained with BODIPY 493/503 (green); lower, HA-Sec8 (red) and lipid droplets (green). Scale = 10 μm.

    Article Snippet: About 50,000 cells/well/100 μL 3T3-L1 adipocytes were plated onto an optical 96 well plate (Fischer Scientific) and centrifuged at 1000 rpm for 5 min. After overnight incubation at 37°C with 5% CO2 , media were changed to serum-free DMEM of high-glucose (25 mM) or low-glucose concentration (5.5 mM), and incubated for additional 1 hour.

    Techniques: Expressing, Staining

    Ceramide and triacylglycerol levels in treated 3T3-L1 adipocytes. (A) Ceramide levels were measured with a monoclonal antibody assay in extracts from adipocytes 24 h after addition of 50 μM (gray bars) or 100 μM (white bars) LA or t 10 c 12 CLA (CLA) with or without 30μM C2 ceramide (C2) or 10μg/ml of an inhibitor of sphingosine kinase (SKI-II), which is expected to increase ceramide levels. (B) Triacylglycerol (TG) levels 24 h after 3T3-L1 adipocytes were treated with 50 μM LA or t 10 c 12 CLA (CLA), with or without 30 μM C2 ceramide or 10 μg/ml SKI-II. The control sample contained 0.2% DMSO, which produced the same values as a control sample containing 0.2% ethanol in separate experiments (data not shown). (C) Triacylglycerol levels 24 h after 3T3-L1 adipocytes were treated 100 μM LA or t 10 c 12 CLA (CLA), with or without 20 μM fumonisin B1 (FB1) or 50 μM myriocin (Myri.). (D) Ceramide levels were detected and quantitated as in A for adipocytes treated for 24 h with 100 μM LA or t 10 c 12 CLA, with or without 20 μM FB1 or 50 μM myriocin. Each bar represents the mean ± SEM (n = 3), and a representative experiment of at least three independent experiments is shown. Means not sharing a common letter differ, p ≤0.05.

    Journal: PLoS ONE

    Article Title: Sphingolipids Are Required for Efficient Triacylglycerol Loss in Conjugated Linoleic Acid Treated Adipocytes

    doi: 10.1371/journal.pone.0119005

    Figure Lengend Snippet: Ceramide and triacylglycerol levels in treated 3T3-L1 adipocytes. (A) Ceramide levels were measured with a monoclonal antibody assay in extracts from adipocytes 24 h after addition of 50 μM (gray bars) or 100 μM (white bars) LA or t 10 c 12 CLA (CLA) with or without 30μM C2 ceramide (C2) or 10μg/ml of an inhibitor of sphingosine kinase (SKI-II), which is expected to increase ceramide levels. (B) Triacylglycerol (TG) levels 24 h after 3T3-L1 adipocytes were treated with 50 μM LA or t 10 c 12 CLA (CLA), with or without 30 μM C2 ceramide or 10 μg/ml SKI-II. The control sample contained 0.2% DMSO, which produced the same values as a control sample containing 0.2% ethanol in separate experiments (data not shown). (C) Triacylglycerol levels 24 h after 3T3-L1 adipocytes were treated 100 μM LA or t 10 c 12 CLA (CLA), with or without 20 μM fumonisin B1 (FB1) or 50 μM myriocin (Myri.). (D) Ceramide levels were detected and quantitated as in A for adipocytes treated for 24 h with 100 μM LA or t 10 c 12 CLA, with or without 20 μM FB1 or 50 μM myriocin. Each bar represents the mean ± SEM (n = 3), and a representative experiment of at least three independent experiments is shown. Means not sharing a common letter differ, p ≤0.05.

    Article Snippet: Metabolic profiling Metabolite profiling of 3T3-L1 adipocytes was performed by Metabolon (Durham, NC).

    Techniques: Produced

    Inhibition of both biosynthetic entry points into ceramides affects triacylglycerol and ceramide levels. (A) Immunoblot analysis of ceramide levels 24 h after 3T3-L1 adipocytes were treated with 100 μM LA or t 10 c 12 CLA in the presence of 100 nM shRNA against non-target (sh-Non) or S1PP (sh-S1PP), with or without 20 μM Fumonisin B1 (FB1) or 50 μM Myriocin (Myri). (B) Triacylglycerol (TG) levels after 3T3-L1 adipocytes were treated as in A. A representative experiment of at least three independent experiments is shown for each panel. Each bar represents the mean ± SEM (n = 3). Means not sharing a common letter differ, p ≤0.05.

    Journal: PLoS ONE

    Article Title: Sphingolipids Are Required for Efficient Triacylglycerol Loss in Conjugated Linoleic Acid Treated Adipocytes

    doi: 10.1371/journal.pone.0119005

    Figure Lengend Snippet: Inhibition of both biosynthetic entry points into ceramides affects triacylglycerol and ceramide levels. (A) Immunoblot analysis of ceramide levels 24 h after 3T3-L1 adipocytes were treated with 100 μM LA or t 10 c 12 CLA in the presence of 100 nM shRNA against non-target (sh-Non) or S1PP (sh-S1PP), with or without 20 μM Fumonisin B1 (FB1) or 50 μM Myriocin (Myri). (B) Triacylglycerol (TG) levels after 3T3-L1 adipocytes were treated as in A. A representative experiment of at least three independent experiments is shown for each panel. Each bar represents the mean ± SEM (n = 3). Means not sharing a common letter differ, p ≤0.05.

    Article Snippet: Metabolic profiling Metabolite profiling of 3T3-L1 adipocytes was performed by Metabolon (Durham, NC).

    Techniques: Inhibition, shRNA

    Treatment of 3T3-L1 adipocytes with BoNT D blocks insulin-stimulated GLUT4–vesicle translocation to the plasma membrane. ( A ) SLO-permeabilized 3T3-L1 adipocytes were pretreated for 20 min with buffer alone (control) or buffer containing 100 nM BoNT D or BoNT C, followed by an additional 10-min incubation with insulin. Plasma membrane sheets were then prepared and GLUT4 was detected by immunofluorescence. ( B ) Following treatment of permeabilized cells with buffer alone (−) or BoNT D (+), an LDM subcellular membrane fraction was prepared and LDM-resident proteins were examined by immunoblot analysis using antisera against the proteins indicated.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

    doi:

    Figure Lengend Snippet: Treatment of 3T3-L1 adipocytes with BoNT D blocks insulin-stimulated GLUT4–vesicle translocation to the plasma membrane. ( A ) SLO-permeabilized 3T3-L1 adipocytes were pretreated for 20 min with buffer alone (control) or buffer containing 100 nM BoNT D or BoNT C, followed by an additional 10-min incubation with insulin. Plasma membrane sheets were then prepared and GLUT4 was detected by immunofluorescence. ( B ) Following treatment of permeabilized cells with buffer alone (−) or BoNT D (+), an LDM subcellular membrane fraction was prepared and LDM-resident proteins were examined by immunoblot analysis using antisera against the proteins indicated.

    Article Snippet: Using an antibody to SNAP-23 we have detected this protein in membrane preparations from 3T3-L1 adipocytes (P. Wong, W. Trimble, A.K., M. Wilson, P. Roche, and B.C., unpublished results).

    Techniques: Translocation Assay, Incubation, Immunofluorescence

    ( A ) Syntaxin-4 is present in 3T3-L1 adipocytes and associates with VAMP-2. Detergent extracts of 3T3-L1 adipocytes were subjected to immunoprecipitation (IP) with a nonimmune antiserum (NI) or anti-syntaxin-4 (αSynt4). The immunoprecipitated proteins were separated by SDS/PAGE and immunoblotted (IB) with either αSynt-4 or αVAMP-2. ( B ) Effect of various GST-fusion proteins on insulin-regulated GLUT4 translocation. Permeabilized adipocytes were incubated in buffer alone or in the presence of 10 μM GST alone, GST–VAMP-2, or GST–syntaxin-4 for 10 min. The cells were then stimulated with insulin for an additional 10 min followed by analysis of GLUT4 translocation by immunofluorescence of plasma membrane sheets.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

    doi:

    Figure Lengend Snippet: ( A ) Syntaxin-4 is present in 3T3-L1 adipocytes and associates with VAMP-2. Detergent extracts of 3T3-L1 adipocytes were subjected to immunoprecipitation (IP) with a nonimmune antiserum (NI) or anti-syntaxin-4 (αSynt4). The immunoprecipitated proteins were separated by SDS/PAGE and immunoblotted (IB) with either αSynt-4 or αVAMP-2. ( B ) Effect of various GST-fusion proteins on insulin-regulated GLUT4 translocation. Permeabilized adipocytes were incubated in buffer alone or in the presence of 10 μM GST alone, GST–VAMP-2, or GST–syntaxin-4 for 10 min. The cells were then stimulated with insulin for an additional 10 min followed by analysis of GLUT4 translocation by immunofluorescence of plasma membrane sheets.

    Article Snippet: Using an antibody to SNAP-23 we have detected this protein in membrane preparations from 3T3-L1 adipocytes (P. Wong, W. Trimble, A.K., M. Wilson, P. Roche, and B.C., unpublished results).

    Techniques: Immunoprecipitation, SDS Page, Translocation Assay, Incubation, Immunofluorescence

    Adipocyte-derived molecules that support eosinophil migration and survival account for eosinophil infiltration in the perigonadal adipose tissue of high fat diet (HFD) fed mice. ( a ) mRNA expression of CC chemokine ligand 11 ( Ccl11 ) and Ccl24 in adipocyte-differentiated 3T3-L1 cells. Representative blots from 3 independent experiments. The full-length blots are presented in Fig. S6 . *** p

    Journal: Scientific Reports

    Article Title: Eosinophils support adipocyte maturation and promote glucose tolerance in obesity

    doi: 10.1038/s41598-018-28371-4

    Figure Lengend Snippet: Adipocyte-derived molecules that support eosinophil migration and survival account for eosinophil infiltration in the perigonadal adipose tissue of high fat diet (HFD) fed mice. ( a ) mRNA expression of CC chemokine ligand 11 ( Ccl11 ) and Ccl24 in adipocyte-differentiated 3T3-L1 cells. Representative blots from 3 independent experiments. The full-length blots are presented in Fig. S6 . *** p

    Article Snippet: Oil Red O staining To evaluate lipid accumulation, adipocyte-differentiated 3T3-L1 cells were stained with Oil Red O (Sigma-Aldrich).

    Techniques: Derivative Assay, Migration, Mouse Assay, Expressing

    Effect of interferon (IFN)-γ and interleukin (IL)-4 treatment on 3T3-L1 adipogenesis. ( a ) 3T3-L1 cells were treated with insulin to differentiate them into adipocytes in the presence of either IFN-γ or IL-4 and stained with Oil Red O. Original magnification × 20. ( b ) Oil Red O in the adipocyte-differentiated 3T3-L1 cells was eluted using isopropanol and the optical density (OD) of the eluate was analysed. * p

    Journal: Scientific Reports

    Article Title: Eosinophils support adipocyte maturation and promote glucose tolerance in obesity

    doi: 10.1038/s41598-018-28371-4

    Figure Lengend Snippet: Effect of interferon (IFN)-γ and interleukin (IL)-4 treatment on 3T3-L1 adipogenesis. ( a ) 3T3-L1 cells were treated with insulin to differentiate them into adipocytes in the presence of either IFN-γ or IL-4 and stained with Oil Red O. Original magnification × 20. ( b ) Oil Red O in the adipocyte-differentiated 3T3-L1 cells was eluted using isopropanol and the optical density (OD) of the eluate was analysed. * p

    Article Snippet: Oil Red O staining To evaluate lipid accumulation, adipocyte-differentiated 3T3-L1 cells were stained with Oil Red O (Sigma-Aldrich).

    Techniques: Staining