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    ATCC mature adipocytes 3t3 l1 preadipocytes
    The components IPTP and TPP induce endogenous expression and recruitment of PPARγ to the aP2 enhancer region. <t>3T3-L1</t> preadipocytes cells exposed to the differentiation cocktail for 6 days were used for the ChIP assay to measure the endogenous expression and recruitment of PPARγ to the aP2 enhancer region by RT-PCR. Positive controls dexamethasone (DEX) and troglitazone (TROG) were used and depicted in ( A ). ( B ) PPARγ recruitment to the aP2 enhancer region after exposure to 20 μM TPP and 10 μM IPTP was determined. Data represents the mean of n = 6 independent replicates ± SEM. Data were relative to 100% input DNA and significance was determined relative to control PPARγ recruitment using a one-way ANOVA followed by Tukey’s post-hoc analysis (* P
    Mature Adipocytes 3t3 L1 Preadipocytes, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mature 3t3 l1 adipocytes
    Effect of ABG extract on glycerol release in mature <t>3T3-L1</t> adipocytes. Extracellular free glycerol was measured with a glycerol quantification kit. Values are expressed as mean ± SD of three separate experiments. ** p
    Mature 3t3 L1 Adipocytes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Lonza mature 3t3 l1 adipocytes
    Oxygen Consumption Rate Following Isoproterenol Treatment. The Seahorse XF Cell Mito Stress Test was performed on mature <t>3T3-L1</t> adipocytes following a 6 (A) or 48 hour (B) treatment with varying doses of isoproterenol (ISO). The last of 3 measurements was used to determine percentage of change in oxygen consumption rate (OCR) within groups (n = 6 per treatment). Coupling efficiency was determined by calculating the percentage of OCR following oligomycin treatment from baseline. Statistical differences compared to control are indicated with * p
    Mature 3t3 L1 Adipocytes, supplied by Lonza, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    The components IPTP and TPP induce endogenous expression and recruitment of PPARγ to the aP2 enhancer region. 3T3-L1 preadipocytes cells exposed to the differentiation cocktail for 6 days were used for the ChIP assay to measure the endogenous expression and recruitment of PPARγ to the aP2 enhancer region by RT-PCR. Positive controls dexamethasone (DEX) and troglitazone (TROG) were used and depicted in ( A ). ( B ) PPARγ recruitment to the aP2 enhancer region after exposure to 20 μM TPP and 10 μM IPTP was determined. Data represents the mean of n = 6 independent replicates ± SEM. Data were relative to 100% input DNA and significance was determined relative to control PPARγ recruitment using a one-way ANOVA followed by Tukey’s post-hoc analysis (* P

    Journal: PLoS ONE

    Article Title: Firemaster® 550 and its components isopropylated triphenyl phosphate and triphenyl phosphate enhance adipogenesis and transcriptional activity of peroxisome proliferator activated receptor (Pparγ) on the adipocyte protein 2 (aP2) promoter

    doi: 10.1371/journal.pone.0175855

    Figure Lengend Snippet: The components IPTP and TPP induce endogenous expression and recruitment of PPARγ to the aP2 enhancer region. 3T3-L1 preadipocytes cells exposed to the differentiation cocktail for 6 days were used for the ChIP assay to measure the endogenous expression and recruitment of PPARγ to the aP2 enhancer region by RT-PCR. Positive controls dexamethasone (DEX) and troglitazone (TROG) were used and depicted in ( A ). ( B ) PPARγ recruitment to the aP2 enhancer region after exposure to 20 μM TPP and 10 μM IPTP was determined. Data represents the mean of n = 6 independent replicates ± SEM. Data were relative to 100% input DNA and significance was determined relative to control PPARγ recruitment using a one-way ANOVA followed by Tukey’s post-hoc analysis (* P

    Article Snippet: Differentiation of 3T3-L1 preadipocytes into mature adipocytes 3T3-L1 preadipocytes (ATCC® CL-173™ ) purchased from the American Type Culture Collection were differentiated as previously described [ ].

    Techniques: Expressing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Temporal expression of transcription factors and terminal differentiation markers after exposure to Firemaster ® 550 and its components. mRNA expression levels were determined in 3T3-L1 preadipocytes 2, 4, 6, and 9 days post-treatment with the differentiation cocktail consisting of IBMX, insulin, and either 100 μM FM550, 10 μM IPTP, 20 μM TPP, or 20 μM TBPH. After the indicated time-points, RNA was extracted and reverse transcribed, and we measured the levels of ( A ) Pparγ , ( B ) Cebpα , ( C ) aP2 , ( D ) Lpl , and ( E ) Plin , which were normalized to β-actin levels and relative to time-matched vehicle control (MI). Data represent the mean ± S.E.M ( n = 3–5). * P

    Journal: PLoS ONE

    Article Title: Firemaster® 550 and its components isopropylated triphenyl phosphate and triphenyl phosphate enhance adipogenesis and transcriptional activity of peroxisome proliferator activated receptor (Pparγ) on the adipocyte protein 2 (aP2) promoter

    doi: 10.1371/journal.pone.0175855

    Figure Lengend Snippet: Temporal expression of transcription factors and terminal differentiation markers after exposure to Firemaster ® 550 and its components. mRNA expression levels were determined in 3T3-L1 preadipocytes 2, 4, 6, and 9 days post-treatment with the differentiation cocktail consisting of IBMX, insulin, and either 100 μM FM550, 10 μM IPTP, 20 μM TPP, or 20 μM TBPH. After the indicated time-points, RNA was extracted and reverse transcribed, and we measured the levels of ( A ) Pparγ , ( B ) Cebpα , ( C ) aP2 , ( D ) Lpl , and ( E ) Plin , which were normalized to β-actin levels and relative to time-matched vehicle control (MI). Data represent the mean ± S.E.M ( n = 3–5). * P

    Article Snippet: Differentiation of 3T3-L1 preadipocytes into mature adipocytes 3T3-L1 preadipocytes (ATCC® CL-173™ ) purchased from the American Type Culture Collection were differentiated as previously described [ ].

    Techniques: Expressing

    Lipid accumulation after exposure to Firemaster ® 550 and its components using 3T3-L1 preadipocytes. ( A ) Preadipocytes were differentiated as described in the Materials and Methods with vehicle with IBMX and insulin (MI) or increasing amounts of Firemaster ® 550 (0.1–200 μM) or with the FM550 components ( B ) IPTP, TPP, TBPH, TBB, and DBB (0.1–20 μM) and ( C ) 250 nM dexamethasone (MID), or 5 μM troglitazone (MIT). After 9 days lipid accumulation was visualized using Nile Red staining and then quantified. Lipid accumulation was normalized to DAPI staining and relative to vehicle-treated cells. The photographs are representative data obtained from experiments run in parallel with all the chemicals. Data represent mean ± SEM for n = 3–5 independent experiments performed in triplicate. Statistical significance * P

    Journal: PLoS ONE

    Article Title: Firemaster® 550 and its components isopropylated triphenyl phosphate and triphenyl phosphate enhance adipogenesis and transcriptional activity of peroxisome proliferator activated receptor (Pparγ) on the adipocyte protein 2 (aP2) promoter

    doi: 10.1371/journal.pone.0175855

    Figure Lengend Snippet: Lipid accumulation after exposure to Firemaster ® 550 and its components using 3T3-L1 preadipocytes. ( A ) Preadipocytes were differentiated as described in the Materials and Methods with vehicle with IBMX and insulin (MI) or increasing amounts of Firemaster ® 550 (0.1–200 μM) or with the FM550 components ( B ) IPTP, TPP, TBPH, TBB, and DBB (0.1–20 μM) and ( C ) 250 nM dexamethasone (MID), or 5 μM troglitazone (MIT). After 9 days lipid accumulation was visualized using Nile Red staining and then quantified. Lipid accumulation was normalized to DAPI staining and relative to vehicle-treated cells. The photographs are representative data obtained from experiments run in parallel with all the chemicals. Data represent mean ± SEM for n = 3–5 independent experiments performed in triplicate. Statistical significance * P

    Article Snippet: Differentiation of 3T3-L1 preadipocytes into mature adipocytes 3T3-L1 preadipocytes (ATCC® CL-173™ ) purchased from the American Type Culture Collection were differentiated as previously described [ ].

    Techniques: Staining

    The effects of selective PPARγ antagonist GW9662 on aP2 mRNA expression and lipid accumulation in the presence of troglitazone, TPP, IPTP, and TBPH. Murine 3T3-L1 preadipocytes were induced to differentiate in the presence of 500 μM IBMX (M), 100 nM insulin (I), supplemented with indicated treatments: 5 μM troglitazone (TROG), 0.25 μM dexamethasone (DEX), 20 μM TPP, 10 μM IPTP, or 20 μM TBPH with either solvent control (Veh) or PPARγ inhibitor GW9662 (GW) twice daily. ( A ) At day 6 of differentiation mRNA expression levels of aP2 were quantified by RT-qPCR normalized to MI control conditions. ( B-E ) At days 2, 4, 6, and 9 lipid accumulation was visualized using Nile Red staining and then quantified. Lipid accumulation was normalized to DAPI staining. Data represent mean ± SEM for n = 3 independent experiments. * denotes p

    Journal: PLoS ONE

    Article Title: Firemaster® 550 and its components isopropylated triphenyl phosphate and triphenyl phosphate enhance adipogenesis and transcriptional activity of peroxisome proliferator activated receptor (Pparγ) on the adipocyte protein 2 (aP2) promoter

    doi: 10.1371/journal.pone.0175855

    Figure Lengend Snippet: The effects of selective PPARγ antagonist GW9662 on aP2 mRNA expression and lipid accumulation in the presence of troglitazone, TPP, IPTP, and TBPH. Murine 3T3-L1 preadipocytes were induced to differentiate in the presence of 500 μM IBMX (M), 100 nM insulin (I), supplemented with indicated treatments: 5 μM troglitazone (TROG), 0.25 μM dexamethasone (DEX), 20 μM TPP, 10 μM IPTP, or 20 μM TBPH with either solvent control (Veh) or PPARγ inhibitor GW9662 (GW) twice daily. ( A ) At day 6 of differentiation mRNA expression levels of aP2 were quantified by RT-qPCR normalized to MI control conditions. ( B-E ) At days 2, 4, 6, and 9 lipid accumulation was visualized using Nile Red staining and then quantified. Lipid accumulation was normalized to DAPI staining. Data represent mean ± SEM for n = 3 independent experiments. * denotes p

    Article Snippet: Differentiation of 3T3-L1 preadipocytes into mature adipocytes 3T3-L1 preadipocytes (ATCC® CL-173™ ) purchased from the American Type Culture Collection were differentiated as previously described [ ].

    Techniques: Expressing, Quantitative RT-PCR, Staining

    SENP1 deletion alters pancreatic adipocyte phenotype and augments NF-κB-dependent inflammation. ( a , b ) Pancreatic adipose was collected from 7 and 14 weeks old Ctrl and SENP1-aP2KO male (male, n =6). Morphology was visualized by haematoxylin and eosin stain staining. Scale bar, 20 μm ( a ). Cell sizes were quantified in ( b ). Three sections from each adipose tissue. Data are representative for three independent experiments. ( c ) Transcript levels of adipocyte differentiation markers (fatty acid synthase, adipose triglyceride lipase and lipoprotein lipase) in PATs were quantified by quantitative PCR with reverse transcription with GAPDH for normalization. n =6, male for each group. ( d ) Increased IKK-NF-κB activities in SENP1-aP2KO PATs. A representative western blot was from Ctrl and SENP1-aP2KO mice at the age of 7 weeks. Data are representative from three independent experiments is shown ( n =6, male). Relative protein levels were quantified from three blots by taking Ctrl as 1.0 ( n =6, male). ( e – h ) NF-κB activation is specifically detected in PATs of SENP1-aP2KO mice. ( e ) Phosphor-p65 staining (red) in PATs but not in pancreas. ( f ) Co-immunostaining of phosphor-p65 (green) and adipocyte marker FABP4 (red). ( g ) High-power images show co-staining of phosphor-p65 (red) in the nucleus of FABP4 + adipocytes (green). ( h ) Co-staining of phosphor-p65 (green) with APC-conjugated adipocyte marker FABP4 (green; yellow arrowheads), but not with macrophage marker F4/80 (red; white arrowheads). Representative images are from one of three sections from SENP1-aP2KO PATs and n =3 male mice. Scale bar, 20 μm. ( i , j ) ChIP assay. ChIP assays with p65/RelA antibody were performed in the adipocyte isolated from Ctrl ( n =6, male) and SENP1-aP2KO mice ( n =6, male) with an IgG isotype as a control. Bindings of p65/RelA to the IL-6, IL-1β and TNF-α gene promoters were quantified with the ratio of IP/input for each promoter ( j ). ( k , l ) Effects of SENP1 knockdown on NF-κB and cytokine expression in adipocytes. 3T3-L1 adipocytes were transfected with control or SENP1-specific siRNA for 24 h. IKK-NF-κB p65/RelA signalling molecules were detected by western blotting. A representative blot from three experiments is shown and protein levels are quantified by taking Ctrl as 1.0 ( k ). Protein levels of IL-6, TNF-α, IFN-γ and IL-1β levels present in adipocyte cultures were detected with ELISA after 24-h culture ( l ). All data are means±s.e.m. of three independent experiments. The two-tailed Student's paired t -test was used for the statistical analysis. * P

    Journal: Nature Communications

    Article Title: SENP1-mediated NEMO deSUMOylation in adipocytes limits inflammatory responses and type-1 diabetes progression

    doi: 10.1038/ncomms9917

    Figure Lengend Snippet: SENP1 deletion alters pancreatic adipocyte phenotype and augments NF-κB-dependent inflammation. ( a , b ) Pancreatic adipose was collected from 7 and 14 weeks old Ctrl and SENP1-aP2KO male (male, n =6). Morphology was visualized by haematoxylin and eosin stain staining. Scale bar, 20 μm ( a ). Cell sizes were quantified in ( b ). Three sections from each adipose tissue. Data are representative for three independent experiments. ( c ) Transcript levels of adipocyte differentiation markers (fatty acid synthase, adipose triglyceride lipase and lipoprotein lipase) in PATs were quantified by quantitative PCR with reverse transcription with GAPDH for normalization. n =6, male for each group. ( d ) Increased IKK-NF-κB activities in SENP1-aP2KO PATs. A representative western blot was from Ctrl and SENP1-aP2KO mice at the age of 7 weeks. Data are representative from three independent experiments is shown ( n =6, male). Relative protein levels were quantified from three blots by taking Ctrl as 1.0 ( n =6, male). ( e – h ) NF-κB activation is specifically detected in PATs of SENP1-aP2KO mice. ( e ) Phosphor-p65 staining (red) in PATs but not in pancreas. ( f ) Co-immunostaining of phosphor-p65 (green) and adipocyte marker FABP4 (red). ( g ) High-power images show co-staining of phosphor-p65 (red) in the nucleus of FABP4 + adipocytes (green). ( h ) Co-staining of phosphor-p65 (green) with APC-conjugated adipocyte marker FABP4 (green; yellow arrowheads), but not with macrophage marker F4/80 (red; white arrowheads). Representative images are from one of three sections from SENP1-aP2KO PATs and n =3 male mice. Scale bar, 20 μm. ( i , j ) ChIP assay. ChIP assays with p65/RelA antibody were performed in the adipocyte isolated from Ctrl ( n =6, male) and SENP1-aP2KO mice ( n =6, male) with an IgG isotype as a control. Bindings of p65/RelA to the IL-6, IL-1β and TNF-α gene promoters were quantified with the ratio of IP/input for each promoter ( j ). ( k , l ) Effects of SENP1 knockdown on NF-κB and cytokine expression in adipocytes. 3T3-L1 adipocytes were transfected with control or SENP1-specific siRNA for 24 h. IKK-NF-κB p65/RelA signalling molecules were detected by western blotting. A representative blot from three experiments is shown and protein levels are quantified by taking Ctrl as 1.0 ( k ). Protein levels of IL-6, TNF-α, IFN-γ and IL-1β levels present in adipocyte cultures were detected with ELISA after 24-h culture ( l ). All data are means±s.e.m. of three independent experiments. The two-tailed Student's paired t -test was used for the statistical analysis. * P

    Article Snippet: Cell lines and isolation of mature adipocytes 3T3-L1, a commonly used preadipocyte mouse cell line, was obtained from ATCC (CL-173) and cultured under DMEM media supplemented with 2 mM glutamine and 10% calf serum.

    Techniques: H&E Stain, Staining, Real-time Polymerase Chain Reaction, Western Blot, Mouse Assay, Activation Assay, Immunostaining, Marker, Chromatin Immunoprecipitation, Isolation, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    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

    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

    HO-1 induction alters macrophage polarization in cocultures. Raw264.7 macrophages were seed onto 3T3-L1 adipocytes with/without being pretreated with the indicated concentrations of hemin or CORM-2 for 12 h. (a) IL-4 levels in hemin/CORM-2-treated cocultures. (b) Total RNAs were isolated and the level of IL-4 was analyzed by qRT-PCR. Transcript levels of the M2 markers Mrc1 (c) and Clec10a (d) and the M1 marker (CD274) (e) were detected by qRT-PCR; β -actin was used as control gene. The experiment was performed in duplicate. Results are means ± SEM. * P

    Journal: Mediators of Inflammation

    Article Title: Induction of Heme Oxygenase-1 with Hemin Reduces Obesity-Induced Adipose Tissue Inflammation via Adipose Macrophage Phenotype Switching

    doi: 10.1155/2014/290708

    Figure Lengend Snippet: HO-1 induction alters macrophage polarization in cocultures. Raw264.7 macrophages were seed onto 3T3-L1 adipocytes with/without being pretreated with the indicated concentrations of hemin or CORM-2 for 12 h. (a) IL-4 levels in hemin/CORM-2-treated cocultures. (b) Total RNAs were isolated and the level of IL-4 was analyzed by qRT-PCR. Transcript levels of the M2 markers Mrc1 (c) and Clec10a (d) and the M1 marker (CD274) (e) were detected by qRT-PCR; β -actin was used as control gene. The experiment was performed in duplicate. Results are means ± SEM. * P

    Article Snippet: Preparation of Adipocyte/Macrophage-Conditioned Medium Adipocyte-conditioned medium was collected from 6-day matured 3T3-L1 adipocytes which were cultured in serum-free medium for 24 h. To prepare Raw264.7 macrophage-conditioned medium, macrophages were incubated for 24 h with 5 μ g/mL lipopolysaccharide (Sigma, St. Louis, MO, USA), washed once with PBS, and cultured in serum-free medium for another 24 h. These conditioned media were collected and filtered to remove debris.

    Techniques: Isolation, Quantitative RT-PCR, Marker

    HO-1 induction in adipocyte and macrophage cocultures. Coculture of 3T3-L1 adipocytes and Raw264.7 macrophages were cocultured in direct contact in a ratio 1 : 1 for 24 h in the presence or absence of hemin (10 μ M). As a control, adipocytes and macrophages were also cultured separately, with cell numbers per well equal to those in the contact system and mixed after harvesting. (a) Total RNAs were isolated and the level of HO-1 transcripts was analyzed by qRT-PCR. 36B4 was used as a control gene. A.U.: arbitrary units. (b) After 24 h of coculture, the cell types were separated using CD11b microbeads. Levels of HO-1 protein expression were measured in coculture, isolated adipocytes (Iso-Adi), and macrophages (Iso-M Ф ) by western blotting; β -actin was used as a control. The experiment was isolated in duplicate. Results are means ± SEM. * P

    Journal: Mediators of Inflammation

    Article Title: Induction of Heme Oxygenase-1 with Hemin Reduces Obesity-Induced Adipose Tissue Inflammation via Adipose Macrophage Phenotype Switching

    doi: 10.1155/2014/290708

    Figure Lengend Snippet: HO-1 induction in adipocyte and macrophage cocultures. Coculture of 3T3-L1 adipocytes and Raw264.7 macrophages were cocultured in direct contact in a ratio 1 : 1 for 24 h in the presence or absence of hemin (10 μ M). As a control, adipocytes and macrophages were also cultured separately, with cell numbers per well equal to those in the contact system and mixed after harvesting. (a) Total RNAs were isolated and the level of HO-1 transcripts was analyzed by qRT-PCR. 36B4 was used as a control gene. A.U.: arbitrary units. (b) After 24 h of coculture, the cell types were separated using CD11b microbeads. Levels of HO-1 protein expression were measured in coculture, isolated adipocytes (Iso-Adi), and macrophages (Iso-M Ф ) by western blotting; β -actin was used as a control. The experiment was isolated in duplicate. Results are means ± SEM. * P

    Article Snippet: Preparation of Adipocyte/Macrophage-Conditioned Medium Adipocyte-conditioned medium was collected from 6-day matured 3T3-L1 adipocytes which were cultured in serum-free medium for 24 h. To prepare Raw264.7 macrophage-conditioned medium, macrophages were incubated for 24 h with 5 μ g/mL lipopolysaccharide (Sigma, St. Louis, MO, USA), washed once with PBS, and cultured in serum-free medium for another 24 h. These conditioned media were collected and filtered to remove debris.

    Techniques: Cell Culture, Isolation, Quantitative RT-PCR, Expressing, Western Blot

    HO-1 induction reduces inflammatory responses in adipocytes and macrophage cocultures. ((a)–(c)) Raw264.7 macrophages were seeded onto 3T3-L1 adipocytes with/without being pretreated with the indicated concentrations of hemin or CORM-2 for 24 h. ZnPP and RuCl 3 were used as HO-1 competitive inhibitor and CORM-2 negative control, respectively. As a control, adipocytes and macrophages were cultured separately, with cell numbers per well equal to those in the contact system. TNF- α (a) and IL-6 (b) protein levels were detected by ELISA. Adiponectin mRNA was detected by qRT-PCR (c). The experiment was set up in triplicate. ((d) and (e)) Adipocytes and macrophages were treated with macrophage-conditioned medium (M Ф -CM) and adipocyte-conditioned medium (Adi-CM), respectively, with/without hemin and CORM-2 pretreatment at the indicated concentrations for 1 h. JNK phosphorylation (p-JNK)/JNK and I κ B α were measured by western blotting. Experiment was performed in duplicate. Results are means ± SEM. * P

    Journal: Mediators of Inflammation

    Article Title: Induction of Heme Oxygenase-1 with Hemin Reduces Obesity-Induced Adipose Tissue Inflammation via Adipose Macrophage Phenotype Switching

    doi: 10.1155/2014/290708

    Figure Lengend Snippet: HO-1 induction reduces inflammatory responses in adipocytes and macrophage cocultures. ((a)–(c)) Raw264.7 macrophages were seeded onto 3T3-L1 adipocytes with/without being pretreated with the indicated concentrations of hemin or CORM-2 for 24 h. ZnPP and RuCl 3 were used as HO-1 competitive inhibitor and CORM-2 negative control, respectively. As a control, adipocytes and macrophages were cultured separately, with cell numbers per well equal to those in the contact system. TNF- α (a) and IL-6 (b) protein levels were detected by ELISA. Adiponectin mRNA was detected by qRT-PCR (c). The experiment was set up in triplicate. ((d) and (e)) Adipocytes and macrophages were treated with macrophage-conditioned medium (M Ф -CM) and adipocyte-conditioned medium (Adi-CM), respectively, with/without hemin and CORM-2 pretreatment at the indicated concentrations for 1 h. JNK phosphorylation (p-JNK)/JNK and I κ B α were measured by western blotting. Experiment was performed in duplicate. Results are means ± SEM. * P

    Article Snippet: Preparation of Adipocyte/Macrophage-Conditioned Medium Adipocyte-conditioned medium was collected from 6-day matured 3T3-L1 adipocytes which were cultured in serum-free medium for 24 h. To prepare Raw264.7 macrophage-conditioned medium, macrophages were incubated for 24 h with 5 μ g/mL lipopolysaccharide (Sigma, St. Louis, MO, USA), washed once with PBS, and cultured in serum-free medium for another 24 h. These conditioned media were collected and filtered to remove debris.

    Techniques: Negative Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

    Oxygen Consumption Rate Following Isoproterenol Treatment. The Seahorse XF Cell Mito Stress Test was performed on mature 3T3-L1 adipocytes following a 6 (A) or 48 hour (B) treatment with varying doses of isoproterenol (ISO). The last of 3 measurements was used to determine percentage of change in oxygen consumption rate (OCR) within groups (n = 6 per treatment). Coupling efficiency was determined by calculating the percentage of OCR following oligomycin treatment from baseline. Statistical differences compared to control are indicated with * p

    Journal: PLoS ONE

    Article Title: Isoproterenol Increases Uncoupling, Glycolysis, and Markers of Beiging in Mature 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0138344

    Figure Lengend Snippet: Oxygen Consumption Rate Following Isoproterenol Treatment. The Seahorse XF Cell Mito Stress Test was performed on mature 3T3-L1 adipocytes following a 6 (A) or 48 hour (B) treatment with varying doses of isoproterenol (ISO). The last of 3 measurements was used to determine percentage of change in oxygen consumption rate (OCR) within groups (n = 6 per treatment). Coupling efficiency was determined by calculating the percentage of OCR following oligomycin treatment from baseline. Statistical differences compared to control are indicated with * p

    Article Snippet: Lipid Quantification AdipoRed™ reagent was used to quantify intracellular lipids in mature 3T3-L1 adipocytes according to manufacturer’s instructions (Lonza).

    Techniques:

    Extracellular Acidification Rate Following Isoproterenol Treatment. The Seahorse XF Cell Mito Stress Test was performed on mature 3T3-L1 adipocytes following a 6 (A) or 48 hour (B) treatment with varying doses of isoproterenol (ISO). The last of 3 measurements was used to determine percentage of change in extracellular acidification rate (ECAR) within groups (n = 6 per treatment) and provides a measurement of glycolysis. The metabolic profile for both 6 (A) and 48 hours (B) was determined by plotting ECAR to OCR. The relationship between coupling efficiency with glycolysis (ECAR) was visualized following 6 and 48 hours of isoproterenol treatment by plotting both points against each other (C). Statistical differences compared to control are indicated with * p

    Journal: PLoS ONE

    Article Title: Isoproterenol Increases Uncoupling, Glycolysis, and Markers of Beiging in Mature 3T3-L1 Adipocytes

    doi: 10.1371/journal.pone.0138344

    Figure Lengend Snippet: Extracellular Acidification Rate Following Isoproterenol Treatment. The Seahorse XF Cell Mito Stress Test was performed on mature 3T3-L1 adipocytes following a 6 (A) or 48 hour (B) treatment with varying doses of isoproterenol (ISO). The last of 3 measurements was used to determine percentage of change in extracellular acidification rate (ECAR) within groups (n = 6 per treatment) and provides a measurement of glycolysis. The metabolic profile for both 6 (A) and 48 hours (B) was determined by plotting ECAR to OCR. The relationship between coupling efficiency with glycolysis (ECAR) was visualized following 6 and 48 hours of isoproterenol treatment by plotting both points against each other (C). Statistical differences compared to control are indicated with * p

    Article Snippet: Lipid Quantification AdipoRed™ reagent was used to quantify intracellular lipids in mature 3T3-L1 adipocytes according to manufacturer’s instructions (Lonza).

    Techniques: