3t3 l1  (ATCC)


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    Structured Review

    ATCC 3t3 l1
    STRA6 depletion inhibits adipogenesis, and this effect depends on RARα and the presence of retinol/RBP4. (A) mRNA expression of STRA6 in <t>3T3-L1</t> preadipocytes after electroporation of STRA6 siRNA (day 0); (B) adipocyte conversion was determined
    3t3 L1, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Retinol-Binding Protein 4 and Its Membrane Receptor STRA6 Control Adipogenesis by Regulating Cellular Retinoid Homeostasis and Retinoic Acid Receptor ? Activity"

    Article Title: Retinol-Binding Protein 4 and Its Membrane Receptor STRA6 Control Adipogenesis by Regulating Cellular Retinoid Homeostasis and Retinoic Acid Receptor ? Activity

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00221-13

    STRA6 depletion inhibits adipogenesis, and this effect depends on RARα and the presence of retinol/RBP4. (A) mRNA expression of STRA6 in 3T3-L1 preadipocytes after electroporation of STRA6 siRNA (day 0); (B) adipocyte conversion was determined
    Figure Legend Snippet: STRA6 depletion inhibits adipogenesis, and this effect depends on RARα and the presence of retinol/RBP4. (A) mRNA expression of STRA6 in 3T3-L1 preadipocytes after electroporation of STRA6 siRNA (day 0); (B) adipocyte conversion was determined

    Techniques Used: Expressing, Electroporation

    Apo-RBP4 enhances adipocyte differentiation in a STRA6-dependent manner. (A) 3T3-L1 cells preloaded with retinol were differentiated and treated with apo-RBP4 during the first 4 days of differentiation, as depicted. Adipocyte conversion was evaluated
    Figure Legend Snippet: Apo-RBP4 enhances adipocyte differentiation in a STRA6-dependent manner. (A) 3T3-L1 cells preloaded with retinol were differentiated and treated with apo-RBP4 during the first 4 days of differentiation, as depicted. Adipocyte conversion was evaluated

    Techniques Used:

    STRA6 overexpression promotes adipocyte differentiation of precursor cells. (A) mRNA and protein expression of retrovirally expressed STRA6 in 3T3-L1 cells at day 0. (B) Adipocyte conversion was assessed 7 days later by phase-contrast microscopy and oil
    Figure Legend Snippet: STRA6 overexpression promotes adipocyte differentiation of precursor cells. (A) mRNA and protein expression of retrovirally expressed STRA6 in 3T3-L1 cells at day 0. (B) Adipocyte conversion was assessed 7 days later by phase-contrast microscopy and oil

    Techniques Used: Over Expression, Expressing, Microscopy

    STRA6 is expressed in 3T3-L1 cells, and holo-RBP4 blocks adipogenesis via RARα. mRNA (A) and protein (B, left) expression of PPARγ and STRA6 before and after the differentiation of 3T3-L1 cells (day 0 and day 7) and in collagenase-digested
    Figure Legend Snippet: STRA6 is expressed in 3T3-L1 cells, and holo-RBP4 blocks adipogenesis via RARα. mRNA (A) and protein (B, left) expression of PPARγ and STRA6 before and after the differentiation of 3T3-L1 cells (day 0 and day 7) and in collagenase-digested

    Techniques Used: Expressing

    STRA6 and RBP4 in precursor cells work independently of STAT5 signaling by regulating retinoid homeostasis and RARα activity. (A) Confluent 3T3-L1 preadipocytes with control or STRA6 overexpression were stimulated for 20 min with GH (500 ng/ml)
    Figure Legend Snippet: STRA6 and RBP4 in precursor cells work independently of STAT5 signaling by regulating retinoid homeostasis and RARα activity. (A) Confluent 3T3-L1 preadipocytes with control or STRA6 overexpression were stimulated for 20 min with GH (500 ng/ml)

    Techniques Used: Activity Assay, Over Expression

    2) Product Images from "In vitro anti-inflammatory and anti-lipid accumulation properties of taxifolin-rich extract from the Japanese larch, Larix kaempferi"

    Article Title: In vitro anti-inflammatory and anti-lipid accumulation properties of taxifolin-rich extract from the Japanese larch, Larix kaempferi

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2020.e05505

    Effects of LK-ME on lipid accumulation in adipocyte differentiated 3T3-L1 cells. Adipocyte differentiated 3T3-L1 cells were grown in medium containing the indicated dilutions of LK-ME (A) or indicated concentrations of taxifolin (B), quercetin (C), and luteolin (D). Berberine chloride (E) was used as positive controls. After 3 days, the cells were fixed and the accumulated lipids stained with Oil Red O. Then the stained Oil Red O was extracted, and measured by spectrophotometer as described in the Materials and Methods section. Lipid accumulations are shown as percent versus lipid accumulation in the untreated control cells after normalization with the protein concentration of cell lysate. The taxifolin concentrations at the indicated dilution of LK-ME are given in parentheses. Error bars indicate standard deviation calculated from three independent experiments. The asterisks (∗) and the double asterisks (∗∗) indicate that the difference is larger than two-fold compared with that of the control, and statistically significant; ∗: p
    Figure Legend Snippet: Effects of LK-ME on lipid accumulation in adipocyte differentiated 3T3-L1 cells. Adipocyte differentiated 3T3-L1 cells were grown in medium containing the indicated dilutions of LK-ME (A) or indicated concentrations of taxifolin (B), quercetin (C), and luteolin (D). Berberine chloride (E) was used as positive controls. After 3 days, the cells were fixed and the accumulated lipids stained with Oil Red O. Then the stained Oil Red O was extracted, and measured by spectrophotometer as described in the Materials and Methods section. Lipid accumulations are shown as percent versus lipid accumulation in the untreated control cells after normalization with the protein concentration of cell lysate. The taxifolin concentrations at the indicated dilution of LK-ME are given in parentheses. Error bars indicate standard deviation calculated from three independent experiments. The asterisks (∗) and the double asterisks (∗∗) indicate that the difference is larger than two-fold compared with that of the control, and statistically significant; ∗: p

    Techniques Used: Staining, Spectrophotometry, Protein Concentration, Standard Deviation

    Effect of LK-ME on cell viability of adipocyte differentiated 3T3-L1 cells. Adipocytes differentiated 3T3-L1 cells were grown in medium containing the indicated dilution of LK-ME (A) or indicated concentration of taxifolin (B), quercetin (C), and luteolin (D). After 3 days, the cell viability was monitored using a Cell Counting Kit-8. The cell viability was expressed as a percentage relative to the unstimulated control cells. The taxifolin concentrations at the indicated dilution of LK-ME are given in parentheses. Error bars indicate standard deviation calculated from three independent experiments. The double asterisks (∗∗) indicate that the cell viability is lower than 80% compared with the control cells, and the difference is statistically significant (p
    Figure Legend Snippet: Effect of LK-ME on cell viability of adipocyte differentiated 3T3-L1 cells. Adipocytes differentiated 3T3-L1 cells were grown in medium containing the indicated dilution of LK-ME (A) or indicated concentration of taxifolin (B), quercetin (C), and luteolin (D). After 3 days, the cell viability was monitored using a Cell Counting Kit-8. The cell viability was expressed as a percentage relative to the unstimulated control cells. The taxifolin concentrations at the indicated dilution of LK-ME are given in parentheses. Error bars indicate standard deviation calculated from three independent experiments. The double asterisks (∗∗) indicate that the cell viability is lower than 80% compared with the control cells, and the difference is statistically significant (p

    Techniques Used: Concentration Assay, Cell Counting, Standard Deviation

    3) Product Images from "Anti-Obesity Potential of Ponciri Fructus: Effects of Extracts, Fractions and Compounds on Adipogenesis in 3T3-L1 Preadipocytes"

    Article Title: Anti-Obesity Potential of Ponciri Fructus: Effects of Extracts, Fractions and Compounds on Adipogenesis in 3T3-L1 Preadipocytes

    Journal: Molecules

    doi: 10.3390/molecules27030676

    Relative expression of adipogenic genes PPAR-γ, SREBP-1, C/EBP-α, aP2, LPL and leptin in differentiated 3T3-L1 cells on treatment with oxypeucedanin at 5 µg/mL and 10 µg/mL concentrations. Results are expressed as mean ± standard deviation of triplicate experiment. Statistical significance difference was checked using two-tailed Student’s t -test. * p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control, ## p ˂ 0.01 vs. normal are used to represent significant difference in expression of gene between the groups.
    Figure Legend Snippet: Relative expression of adipogenic genes PPAR-γ, SREBP-1, C/EBP-α, aP2, LPL and leptin in differentiated 3T3-L1 cells on treatment with oxypeucedanin at 5 µg/mL and 10 µg/mL concentrations. Results are expressed as mean ± standard deviation of triplicate experiment. Statistical significance difference was checked using two-tailed Student’s t -test. * p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control, ## p ˂ 0.01 vs. normal are used to represent significant difference in expression of gene between the groups.

    Techniques Used: Expressing, Standard Deviation, Two Tailed Test

    ( A ) Effect of ethanol extracts, hexane fraction and ethyl acetate fraction on percentage lipid deposition by 3T3-L1 cells using ORO assay. ( B )Lipid accumulation in 3T3-L1 cell observed by EVOS XL microscope at 10× magnification after ORO staining. Each set of data represents mean of triplicate experiment ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test. ** p ˂ 0.01 vs. control, ## p ˂ 0.01 vs. normal are used to represent significant difference in lipid production of the sample compared to non-t reated control group.
    Figure Legend Snippet: ( A ) Effect of ethanol extracts, hexane fraction and ethyl acetate fraction on percentage lipid deposition by 3T3-L1 cells using ORO assay. ( B )Lipid accumulation in 3T3-L1 cell observed by EVOS XL microscope at 10× magnification after ORO staining. Each set of data represents mean of triplicate experiment ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test. ** p ˂ 0.01 vs. control, ## p ˂ 0.01 vs. normal are used to represent significant difference in lipid production of the sample compared to non-t reated control group.

    Techniques Used: Oro Assay, Microscopy, Staining, Standard Deviation, Two Tailed Test

    Cell viability of 3T3-L1 cell line on treatment with ethanol extracts, hexane fraction, ethyl acetate fraction, butanol fraction and water fraction of the Ponciri Fructus. Each set of data represents mean of triplicate experiment ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test. * p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control are used to represent significant difference in cell viability of the extract sample compared to non-treated control group.
    Figure Legend Snippet: Cell viability of 3T3-L1 cell line on treatment with ethanol extracts, hexane fraction, ethyl acetate fraction, butanol fraction and water fraction of the Ponciri Fructus. Each set of data represents mean of triplicate experiment ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test. * p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control are used to represent significant difference in cell viability of the extract sample compared to non-treated control group.

    Techniques Used: Standard Deviation, Two Tailed Test

    ( A )Effect of poncirin, phellopterin and oxypeucedanin treatment on percentage lipid deposition by 3T3-L1 cells using ORO assay. ( B ) Lipid accumulation in 3T3-L1 cell observed by EVOS XL microscope at 10× magnification after ORO staining. Each set of data represents mean of triplicate experiments ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test.* p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control, ## p ˂ 0.01 vs. normal are used to represent significant difference in lipid production of the sample compared to non-treated control group.
    Figure Legend Snippet: ( A )Effect of poncirin, phellopterin and oxypeucedanin treatment on percentage lipid deposition by 3T3-L1 cells using ORO assay. ( B ) Lipid accumulation in 3T3-L1 cell observed by EVOS XL microscope at 10× magnification after ORO staining. Each set of data represents mean of triplicate experiments ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test.* p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control, ## p ˂ 0.01 vs. normal are used to represent significant difference in lipid production of the sample compared to non-treated control group.

    Techniques Used: Oro Assay, Microscopy, Staining, Standard Deviation, Two Tailed Test

    Cell viability of 3T3-L1 cell on treatment with poncirin, naringin, auraptene, β -sitosterol, imperatorin, phellopterin and oxypeucedanin isolated from Ponciri Fructus. Each set of data represents mean of triplicate experiment ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test. * p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control are used to represent significant difference in toxicity of the extract sample compared to non-treated control group.
    Figure Legend Snippet: Cell viability of 3T3-L1 cell on treatment with poncirin, naringin, auraptene, β -sitosterol, imperatorin, phellopterin and oxypeucedanin isolated from Ponciri Fructus. Each set of data represents mean of triplicate experiment ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test. * p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control are used to represent significant difference in toxicity of the extract sample compared to non-treated control group.

    Techniques Used: Isolation, Standard Deviation, Two Tailed Test

    ( A )Expression of PPAR- γ , FABP-4, SREBP-1 and actin proteins in 3T3-L1 cells on treatment with different doses of oxypeucedanin measured by Western blot analysis. ( B ) Densitometry analysis was performed to quantify protein levels and results were presented as mean of ( n = 3) ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test. * p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control are used to represent significant difference in expression of adipogenic proteins in treated group compared to non-treated control group.
    Figure Legend Snippet: ( A )Expression of PPAR- γ , FABP-4, SREBP-1 and actin proteins in 3T3-L1 cells on treatment with different doses of oxypeucedanin measured by Western blot analysis. ( B ) Densitometry analysis was performed to quantify protein levels and results were presented as mean of ( n = 3) ± standard deviation. Significant difference between the groups was calculated using two-tailed Student’s t -test. * p ˂ 0.05 vs. control, ** p ˂ 0.01 vs. control are used to represent significant difference in expression of adipogenic proteins in treated group compared to non-treated control group.

    Techniques Used: Expressing, Western Blot, Standard Deviation, Two Tailed Test

    4) Product Images from "CDK4 is an essential insulin effector in adipocytes"

    Article Title: CDK4 is an essential insulin effector in adipocytes

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI81480

    CDK4 is activated by insulin and translates insulin signaling in adipocytes. ( A ) CDK4 activity in vivo. SDS-PAGE autoradiography showing RB1 phosphorylation by CDK4 immunoprecipitated from 3T3-L1 mature adipocytes after insulin stimulation. The left panel shows RB1 phosphorylation by recombinant CDK4 used as a positive control. ( B ) Western blot analysis showing the inhibition of insulin-induced RB1 phosphorylation on Ser780 by CDK4 knockdown in mature 3T3-L1 adipocytes. ( C ) CCND3 and CDK4 association is increased upon insulin stimulation, but decreased upon cotreatment with insulin and AKT inhibitor in mature 3T3-L1 adipocytes. ( D ) Volcano plot showing differences in putative kinase activities between control and Cdk4 nc mice injected (portal vein) with insulin for 3 minutes ( n = 4). Kinases with a positive kinase statistic show higher activity in Cdk4 nc samples compared with control samples, whereas kinases with negative kinase statistic show lower activity in Cdk4 nc samples compared with control samples. ( E ) Based on the upstream kinase activity ( D ) results and on the GeneGO analysis of the PamGene experiment, the putative role of CDK4 upstream of AKT in the insulin signaling pathway is represented. ( F ) Immunoblot showing AKT phosphorylation on Thr308 and Ser473 in response to insulin injection (3 minutes) in fasted control and Cdk4 nc mice ( n = 2 for NaCl and n = 4 for insulin treatment). ( G and H ) Coimmunoprecipitation experiments showing the interaction between endogenous IRS2 and PIK3R1. IRS2 ( G ) and PIK3R1 ( H ) were immunoprecipitated and the presence of PIK3R1 ( G ) and IRS2 ( H ) was detected by Western blot analysis in Cdk4 +/+ and Cdk4 nc mice treated with insulin for 3 minutes ( n = 2 for NaCl and n = 3 for insulin treatment). ( I and J ) Coimmunoprecipitation experiments showing the interaction between endogenous PIK3R1 or IRS2 and CDK4. CDK4 was immunoprecipitated and the presence of PIK3R1 ( n = 3) ( I ) and IRS2 ( n = 1) ( J ) was detected by Western blot analysis in 3T3-L1 mature adipocytes treated with insulin. A representative Western blot is shown. Unless specified otherwise, all experiments are representative of 3 independent experiments.
    Figure Legend Snippet: CDK4 is activated by insulin and translates insulin signaling in adipocytes. ( A ) CDK4 activity in vivo. SDS-PAGE autoradiography showing RB1 phosphorylation by CDK4 immunoprecipitated from 3T3-L1 mature adipocytes after insulin stimulation. The left panel shows RB1 phosphorylation by recombinant CDK4 used as a positive control. ( B ) Western blot analysis showing the inhibition of insulin-induced RB1 phosphorylation on Ser780 by CDK4 knockdown in mature 3T3-L1 adipocytes. ( C ) CCND3 and CDK4 association is increased upon insulin stimulation, but decreased upon cotreatment with insulin and AKT inhibitor in mature 3T3-L1 adipocytes. ( D ) Volcano plot showing differences in putative kinase activities between control and Cdk4 nc mice injected (portal vein) with insulin for 3 minutes ( n = 4). Kinases with a positive kinase statistic show higher activity in Cdk4 nc samples compared with control samples, whereas kinases with negative kinase statistic show lower activity in Cdk4 nc samples compared with control samples. ( E ) Based on the upstream kinase activity ( D ) results and on the GeneGO analysis of the PamGene experiment, the putative role of CDK4 upstream of AKT in the insulin signaling pathway is represented. ( F ) Immunoblot showing AKT phosphorylation on Thr308 and Ser473 in response to insulin injection (3 minutes) in fasted control and Cdk4 nc mice ( n = 2 for NaCl and n = 4 for insulin treatment). ( G and H ) Coimmunoprecipitation experiments showing the interaction between endogenous IRS2 and PIK3R1. IRS2 ( G ) and PIK3R1 ( H ) were immunoprecipitated and the presence of PIK3R1 ( G ) and IRS2 ( H ) was detected by Western blot analysis in Cdk4 +/+ and Cdk4 nc mice treated with insulin for 3 minutes ( n = 2 for NaCl and n = 3 for insulin treatment). ( I and J ) Coimmunoprecipitation experiments showing the interaction between endogenous PIK3R1 or IRS2 and CDK4. CDK4 was immunoprecipitated and the presence of PIK3R1 ( n = 3) ( I ) and IRS2 ( n = 1) ( J ) was detected by Western blot analysis in 3T3-L1 mature adipocytes treated with insulin. A representative Western blot is shown. Unless specified otherwise, all experiments are representative of 3 independent experiments.

    Techniques Used: Activity Assay, In Vivo, SDS Page, Autoradiography, Immunoprecipitation, Recombinant, Positive Control, Western Blot, Inhibition, Mouse Assay, Injection

    CDK4 phosphorylates in vivo the IRS2 protein at the Ser388. ( A – D ) Immunoblot analysis of IRS2 phosphorylation on Ser388 and AKT phosphorylation on Thr308 and Ser473 in control and Cdk4 nc ( n = 2 starved/5 insulin for Cdk4 +/+ and n = 2 starved/5 insulin for Cdk4 nc ) mice ( A ). ( B ) Quantification of the blot shown in A using ImageJ software. Cdk4 R24C/R24C ( n = 2 starved/3 insulin for both Cdk4 +/+ and Cdk R24C/R24C ). ( C ) Mice were treated for 50 minutes with insulin. ( D ) Quantification of the blot shown in C using ImageJ software. ( E ) Immunoblot analysis of IRS2 phosphorylation on Ser388 and AKT phosphorylation on Thr308 and Ser473 in 3T3-L1 mature adipocytes during a time course insulin stimulation with or without PD0332991 ( n = 1). ( F ) Quantification of the blot shown in E using ImageJ software. ( G ) Correlation between the pIRS2 Ser388/ACTB ratio in VAT and the BMI of the subjects ( n = 45, Pearson’s r = 0.3307, P
    Figure Legend Snippet: CDK4 phosphorylates in vivo the IRS2 protein at the Ser388. ( A – D ) Immunoblot analysis of IRS2 phosphorylation on Ser388 and AKT phosphorylation on Thr308 and Ser473 in control and Cdk4 nc ( n = 2 starved/5 insulin for Cdk4 +/+ and n = 2 starved/5 insulin for Cdk4 nc ) mice ( A ). ( B ) Quantification of the blot shown in A using ImageJ software. Cdk4 R24C/R24C ( n = 2 starved/3 insulin for both Cdk4 +/+ and Cdk R24C/R24C ). ( C ) Mice were treated for 50 minutes with insulin. ( D ) Quantification of the blot shown in C using ImageJ software. ( E ) Immunoblot analysis of IRS2 phosphorylation on Ser388 and AKT phosphorylation on Thr308 and Ser473 in 3T3-L1 mature adipocytes during a time course insulin stimulation with or without PD0332991 ( n = 1). ( F ) Quantification of the blot shown in E using ImageJ software. ( G ) Correlation between the pIRS2 Ser388/ACTB ratio in VAT and the BMI of the subjects ( n = 45, Pearson’s r = 0.3307, P

    Techniques Used: In Vivo, Mouse Assay, Software

    Positive correlation between CDK4 activity and WAT mass. ( A ) Expression levels of CCND1, CCND2, CCND3, CDK4, and CDK6 proteins in mouse eWAT, BAT, brain, muscle, heart, kidney, lung, spleen, and liver. Representative blot of several animals analyzed is shown. ( B ) CDK4 protein level in the SVF and mature adipocytes isolated from VAT. ( C ) Subcellular localization of CCND1, CCND2, CCND3, and CDK4 proteins in cytoplasm and nuclear fractions of eWAT and mature 3T3-L1 adipocytes. LMNA was used as a control for the nuclear fraction. ( B and C ) Representative blots out of 3 independent experiments are shown. ( D and E ) Body weight and percentage of fat mass of 20-week-old Cdk4 +/+ and Cdk4 nc mice ( n = 9) ( D ) and 30-week-old Cdk4 +/+ and Cdk4 R24C/R24C mice ( n = 8) ( E ) as obtained using EchoMRI technology. ( F ) H E staining of eWAT sections from Cdk4 +/+ , Cdk4 nc , and Cdk4 R24C/R24C mice. ( G ) Body weight, Δ fat mass of 20-week-old Cdk4 flox/flox mice infected with AAV8-mini/aP2-null ( n = 5) or AAV8-mini/aP2-cre ( n = 4) analyzed by EchoMRI technology (we show the difference between the percentage of fat before and the percentage of fat 3 weeks after infection). ( H ) H E staining of eWAT sections from Cdk4 flox/flox mice infected with AAV8-mini/aP2-null or AAV8-mini/aP2-cre. ( I ) Body weight and percentage of fat mass of 30-week-old E2f1 +/+ ( n = 4), Cdk4 R24C/R24C E2f1 +/+ ( n = 6), and Cdk4 R24C/R24C E2f1 –/– mice ( n = 12). ( J ) H E staining of eWAT sections from E2f1 +/+ , Cdk4 R24C/R24C E2f1 +/+ , and Cdk4 R24C/R24C E2f1 –/– mice. Statistically significant differences were determined with unpaired 2-tailed Student’s t tests ( D , E , and G ) or 1-way ANOVA followed by Tukey’s multiple comparisons test ( I ). * P
    Figure Legend Snippet: Positive correlation between CDK4 activity and WAT mass. ( A ) Expression levels of CCND1, CCND2, CCND3, CDK4, and CDK6 proteins in mouse eWAT, BAT, brain, muscle, heart, kidney, lung, spleen, and liver. Representative blot of several animals analyzed is shown. ( B ) CDK4 protein level in the SVF and mature adipocytes isolated from VAT. ( C ) Subcellular localization of CCND1, CCND2, CCND3, and CDK4 proteins in cytoplasm and nuclear fractions of eWAT and mature 3T3-L1 adipocytes. LMNA was used as a control for the nuclear fraction. ( B and C ) Representative blots out of 3 independent experiments are shown. ( D and E ) Body weight and percentage of fat mass of 20-week-old Cdk4 +/+ and Cdk4 nc mice ( n = 9) ( D ) and 30-week-old Cdk4 +/+ and Cdk4 R24C/R24C mice ( n = 8) ( E ) as obtained using EchoMRI technology. ( F ) H E staining of eWAT sections from Cdk4 +/+ , Cdk4 nc , and Cdk4 R24C/R24C mice. ( G ) Body weight, Δ fat mass of 20-week-old Cdk4 flox/flox mice infected with AAV8-mini/aP2-null ( n = 5) or AAV8-mini/aP2-cre ( n = 4) analyzed by EchoMRI technology (we show the difference between the percentage of fat before and the percentage of fat 3 weeks after infection). ( H ) H E staining of eWAT sections from Cdk4 flox/flox mice infected with AAV8-mini/aP2-null or AAV8-mini/aP2-cre. ( I ) Body weight and percentage of fat mass of 30-week-old E2f1 +/+ ( n = 4), Cdk4 R24C/R24C E2f1 +/+ ( n = 6), and Cdk4 R24C/R24C E2f1 –/– mice ( n = 12). ( J ) H E staining of eWAT sections from E2f1 +/+ , Cdk4 R24C/R24C E2f1 +/+ , and Cdk4 R24C/R24C E2f1 –/– mice. Statistically significant differences were determined with unpaired 2-tailed Student’s t tests ( D , E , and G ) or 1-way ANOVA followed by Tukey’s multiple comparisons test ( I ). * P

    Techniques Used: Activity Assay, Expressing, Isolation, Mouse Assay, Staining, Infection

    5) Product Images from "Biocompatibility analysis of high molecular weight chitosan obtained from Pleoticus muelleri shrimps. Evaluation in prokaryotic and eukaryotic cells"

    Article Title: Biocompatibility analysis of high molecular weight chitosan obtained from Pleoticus muelleri shrimps. Evaluation in prokaryotic and eukaryotic cells

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2020.100842

    A. Cell proliferation and viability assays for 3T3-L1 fibroblasts under H-CS treatment by means of MTT (i), Crystal violet (ii), TB exclusion (iii) and phase-contrast microscopy (iv) analysis. B. CLSM image showing Cyn-H-CS (50 μg/mL) uptake and the corresponding fluorescence intensity plot in 3D view. * p
    Figure Legend Snippet: A. Cell proliferation and viability assays for 3T3-L1 fibroblasts under H-CS treatment by means of MTT (i), Crystal violet (ii), TB exclusion (iii) and phase-contrast microscopy (iv) analysis. B. CLSM image showing Cyn-H-CS (50 μg/mL) uptake and the corresponding fluorescence intensity plot in 3D view. * p

    Techniques Used: MTT Assay, Microscopy, Confocal Laser Scanning Microscopy, Fluorescence

    6) Product Images from "Fatty acid tryptamide from cacao elongates Drosophila melanogaster lifespan with sirtuin-dependent heat shock protein expression"

    Article Title: Fatty acid tryptamide from cacao elongates Drosophila melanogaster lifespan with sirtuin-dependent heat shock protein expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-022-16471-1

    Effects of Fa-Trp lipid accumulation in HepG2 cells and 3T3-L1 adipocytes. ( a ) HepG2 cells were treated with 300 μmol/L palmitic acid conjugated by bovine serum albumin for 24 h. Oil Red O staining of HepG2 cells with Fa-Trp. The number of cells positive for Oil Red O staining was measured by taking a microscopic image of the cell culture and counting the number of cells stained red per 40-mm2 field. ( b ) Mouse 3T3-L1 cells were differentiated into adipocytes. After 24 h of stimulation with Fa-Trp, Oil Red O staining was performed. The relative optical density (OD) values of mature 3T3-L1 adipocytes were determined at OD540. All values are means of three determinations. Errors = s.e. *p
    Figure Legend Snippet: Effects of Fa-Trp lipid accumulation in HepG2 cells and 3T3-L1 adipocytes. ( a ) HepG2 cells were treated with 300 μmol/L palmitic acid conjugated by bovine serum albumin for 24 h. Oil Red O staining of HepG2 cells with Fa-Trp. The number of cells positive for Oil Red O staining was measured by taking a microscopic image of the cell culture and counting the number of cells stained red per 40-mm2 field. ( b ) Mouse 3T3-L1 cells were differentiated into adipocytes. After 24 h of stimulation with Fa-Trp, Oil Red O staining was performed. The relative optical density (OD) values of mature 3T3-L1 adipocytes were determined at OD540. All values are means of three determinations. Errors = s.e. *p

    Techniques Used: Staining, Cell Culture

    7) Product Images from "Adipocyte-derived extracellular vesicles regulate survival and function of pancreatic β cells"

    Article Title: Adipocyte-derived extracellular vesicles regulate survival and function of pancreatic β cells

    Journal: JCI Insight

    doi: 10.1172/jci.insight.141962

    Effect of Ad-EVs and CK-EVs on survival, proliferation, apoptosis, and function of INS-1E β cells. ( A ) Top: Representative fluorescence microscopy micrographs showing internalization of Ad-EVs in INS-1E β cells, incubated at the indicated times with unlabeled Ad-EVs (Control) or with Ad-EVs labeled with PKH26 (red dye) (scale bar: 10 μm). Bottom: Internalization of Ad-EVs for 24 hours in cells untreated (-CD29) or preincubated for 1 hour with the blocking antibody against CD29 (+CD29). Nuclei were stained blue with DAPI (scale bar: 10 μm). Cell survival ( B ), cell proliferation ( C ), and apoptosis ( D ) assessed by MTT, BrdU, and caspase-3 activity, respectively, in cells cultured in serum-deprived medium for 12 hours, then untreated (control [c]) or treated for a further 24 hours with Ad-EVs (10 × 10 3 /cell) or with EVs from 3T3-L1 adipocytes treated for 24 hours with cytokines (CKs) (CK-EVs) (TNF-α/IFN-γ/IL-1β [50, 25, and 2.5 ng/mL, respectively]). ( E ) Cell survival in cells treated for 24 hours with or without Ad-EVs and anti-CD29 antibody. Results are expressed as percentage of control (mean ± SEM). * P
    Figure Legend Snippet: Effect of Ad-EVs and CK-EVs on survival, proliferation, apoptosis, and function of INS-1E β cells. ( A ) Top: Representative fluorescence microscopy micrographs showing internalization of Ad-EVs in INS-1E β cells, incubated at the indicated times with unlabeled Ad-EVs (Control) or with Ad-EVs labeled with PKH26 (red dye) (scale bar: 10 μm). Bottom: Internalization of Ad-EVs for 24 hours in cells untreated (-CD29) or preincubated for 1 hour with the blocking antibody against CD29 (+CD29). Nuclei were stained blue with DAPI (scale bar: 10 μm). Cell survival ( B ), cell proliferation ( C ), and apoptosis ( D ) assessed by MTT, BrdU, and caspase-3 activity, respectively, in cells cultured in serum-deprived medium for 12 hours, then untreated (control [c]) or treated for a further 24 hours with Ad-EVs (10 × 10 3 /cell) or with EVs from 3T3-L1 adipocytes treated for 24 hours with cytokines (CKs) (CK-EVs) (TNF-α/IFN-γ/IL-1β [50, 25, and 2.5 ng/mL, respectively]). ( E ) Cell survival in cells treated for 24 hours with or without Ad-EVs and anti-CD29 antibody. Results are expressed as percentage of control (mean ± SEM). * P

    Techniques Used: Fluorescence, Microscopy, Incubation, Labeling, Blocking Assay, Staining, MTT Assay, Activity Assay, Cell Culture

    Characterization of EVs released from 3T3-L1 adipocytes and human subcutaneous adipose tissue. Analysis of size distribution, showing the size and number of EVs released from 3T3-L1 adipocytes (Ad-EVs) ( A ) and human subcutaneous adipose tissue (SAT-EVs) ( B ) using the NanoSight technology ( n = 3). Insets show representative micrographs obtained by TEM of purified Ad-EVs and SAT-EVs (scale bar: 100 nm). ( C and D ) Representative Western blot images for EV markers Alix, CD9, and CD63 in 3T3-L1 adipocytes and Ad-EVs ( C ) and in SAT and SAT-EVs ( D ) ( n = 3). ( E and F ) Representative Western blot for adiponectin, leptin, FABP4, and PPARγ in 3T3-L1 cells and Ad-EVs ( E ) and in SAT and SAT-EVs ( F ). Actin served as internal control ( n = 3). ( G and H ) Representative gene expression for adiponectin, leptin, FABP4, PPARγ1, and PPARγ2 in 3T3-L1 and Ad-EVs ( G ) and in SAT and SAT-EVs ( H ), as assessed by RT-PCR. 18s rRNA served as internal control ( n = 3).
    Figure Legend Snippet: Characterization of EVs released from 3T3-L1 adipocytes and human subcutaneous adipose tissue. Analysis of size distribution, showing the size and number of EVs released from 3T3-L1 adipocytes (Ad-EVs) ( A ) and human subcutaneous adipose tissue (SAT-EVs) ( B ) using the NanoSight technology ( n = 3). Insets show representative micrographs obtained by TEM of purified Ad-EVs and SAT-EVs (scale bar: 100 nm). ( C and D ) Representative Western blot images for EV markers Alix, CD9, and CD63 in 3T3-L1 adipocytes and Ad-EVs ( C ) and in SAT and SAT-EVs ( D ) ( n = 3). ( E and F ) Representative Western blot for adiponectin, leptin, FABP4, and PPARγ in 3T3-L1 cells and Ad-EVs ( E ) and in SAT and SAT-EVs ( F ). Actin served as internal control ( n = 3). ( G and H ) Representative gene expression for adiponectin, leptin, FABP4, PPARγ1, and PPARγ2 in 3T3-L1 and Ad-EVs ( G ) and in SAT and SAT-EVs ( H ), as assessed by RT-PCR. 18s rRNA served as internal control ( n = 3).

    Techniques Used: Transmission Electron Microscopy, Purification, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

    8) Product Images from "D609 inhibition of phosphatidylcholine-specific phospholipase C attenuates prolonged insulin stimulation-mediated GLUT4 downregulation in 3T3-L1 adipocytes"

    Article Title: D609 inhibition of phosphatidylcholine-specific phospholipase C attenuates prolonged insulin stimulation-mediated GLUT4 downregulation in 3T3-L1 adipocytes

    Journal: Journal of Physiology and Biochemistry

    doi: 10.1007/s13105-022-00872-x

    Effect of exogenous PC-PLC and its products (PCho and DG) on GLUT4 concentrations. The 3T3-L1 adipocytes were treated with or without the following at indicated concentrations for 4 h: PC-PLC ( a ), PCho ( b ), and DG ( c ). After incubating, the cells were lysed and processed for immunoblotting. The upper panels show the representative GLUT4 immunoblots, and the lower panels display the GLUT4 quantification. The results shown are the mean ± SD ( n = 3)
    Figure Legend Snippet: Effect of exogenous PC-PLC and its products (PCho and DG) on GLUT4 concentrations. The 3T3-L1 adipocytes were treated with or without the following at indicated concentrations for 4 h: PC-PLC ( a ), PCho ( b ), and DG ( c ). After incubating, the cells were lysed and processed for immunoblotting. The upper panels show the representative GLUT4 immunoblots, and the lower panels display the GLUT4 quantification. The results shown are the mean ± SD ( n = 3)

    Techniques Used: Planar Chromatography, Western Blot

    Effect of PC-PLC on H 2 O 2 . Transfection of the 3T3-L1 adipocytes was performed by electroporation with the pHyPer-Cyto expression vector (30 μg), after which the cells were cultured for 24 h on a 35-mm glass dish prior to the fluorescence measurements. The cells were stimulated with indicated concentrations of insulin followed by D609 ( a ), insulin ( b ), PC-PLC ( c ), or PCho ( d ) at the time points indicated with arrows. The data are presented as the mean ± S.E. ( n = 5)
    Figure Legend Snippet: Effect of PC-PLC on H 2 O 2 . Transfection of the 3T3-L1 adipocytes was performed by electroporation with the pHyPer-Cyto expression vector (30 μg), after which the cells were cultured for 24 h on a 35-mm glass dish prior to the fluorescence measurements. The cells were stimulated with indicated concentrations of insulin followed by D609 ( a ), insulin ( b ), PC-PLC ( c ), or PCho ( d ) at the time points indicated with arrows. The data are presented as the mean ± S.E. ( n = 5)

    Techniques Used: Planar Chromatography, Transfection, Electroporation, Expressing, Plasmid Preparation, Cell Culture, Fluorescence

    Effect of insulin stimulation on PC-PLC activity. The 3T3-L1 adipocytes were treated with or without D609 (200 μM) for 30 min and then stimulated with insulin for indicated time intervals. The PC-PLC activity in the membranes ( a ) and total lysate ( b ) were assayed as described in the “ Materials and methods ” section
    Figure Legend Snippet: Effect of insulin stimulation on PC-PLC activity. The 3T3-L1 adipocytes were treated with or without D609 (200 μM) for 30 min and then stimulated with insulin for indicated time intervals. The PC-PLC activity in the membranes ( a ) and total lysate ( b ) were assayed as described in the “ Materials and methods ” section

    Techniques Used: Planar Chromatography, Activity Assay

    Prolonged insulin stimulation requires PC-PLC activity for the downregulation of GLUT4. Prior to stimulation with insulin (500 nM) for 4 h, the 3T3-L1 adipocytes were treated with or without the following compounds at indicated concentrations for 30 min: D609 ( a ), GW4869 ( b ), U73122 ( c ), 1-Butanol ( d ), or HC-3 ( e ). GLUT4 were detected by immunoblotting. The upper panels show the representative immunoblots for GLUT4, and the lower panels were used for quantifying the GLUT4. The results shown are the mean ± SD ( n = 3)
    Figure Legend Snippet: Prolonged insulin stimulation requires PC-PLC activity for the downregulation of GLUT4. Prior to stimulation with insulin (500 nM) for 4 h, the 3T3-L1 adipocytes were treated with or without the following compounds at indicated concentrations for 30 min: D609 ( a ), GW4869 ( b ), U73122 ( c ), 1-Butanol ( d ), or HC-3 ( e ). GLUT4 were detected by immunoblotting. The upper panels show the representative immunoblots for GLUT4, and the lower panels were used for quantifying the GLUT4. The results shown are the mean ± SD ( n = 3)

    Techniques Used: Planar Chromatography, Activity Assay, Western Blot

    9) Product Images from "Intraflagellar-transport A dysfunction causes hyperphagia-induced systemic insulin resistance in a pre-obese state"

    Article Title: Intraflagellar-transport A dysfunction causes hyperphagia-induced systemic insulin resistance in a pre-obese state

    Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    doi: 10.1096/fj.201900751R

    Thm1 knock-down in pre-adipocytes enhances differentiation. A) Western blot analysis for THM1 on extracts of EV and Thm1 kd 3T3–L1 preadipocyte clonal lines. B) Immunostaining for primary cilia markers on EV (P2C3) and Thm1 knock-down cells (P1C1) with quantification. Graphs represent mean ± SD. Statistical significance was determined by T test. **P
    Figure Legend Snippet: Thm1 knock-down in pre-adipocytes enhances differentiation. A) Western blot analysis for THM1 on extracts of EV and Thm1 kd 3T3–L1 preadipocyte clonal lines. B) Immunostaining for primary cilia markers on EV (P2C3) and Thm1 knock-down cells (P1C1) with quantification. Graphs represent mean ± SD. Statistical significance was determined by T test. **P

    Techniques Used: Western Blot, Immunostaining

    Thm1 -deficient 3T3–L1 cells are sensitized to insulin signaling. A) qPCR of pre-adipocytes at DD0. N=5 for EV (P2C3); N=5 for Thm1 .
    Figure Legend Snippet: Thm1 -deficient 3T3–L1 cells are sensitized to insulin signaling. A) qPCR of pre-adipocytes at DD0. N=5 for EV (P2C3); N=5 for Thm1 .

    Techniques Used: Real-time Polymerase Chain Reaction

    Thm1 -deficient 3T3–L1 cells show earlier induction of adipogenesis master transcriptional regulators, C/EBPα and PPARγ. A) Western blot analysis at different differentiation days. B) Immunostaining at DD1 and DD8. Experiments were performed ≥3 times with EV (P2C3) and Thm1 kd cells (P1C1).
    Figure Legend Snippet: Thm1 -deficient 3T3–L1 cells show earlier induction of adipogenesis master transcriptional regulators, C/EBPα and PPARγ. A) Western blot analysis at different differentiation days. B) Immunostaining at DD1 and DD8. Experiments were performed ≥3 times with EV (P2C3) and Thm1 kd cells (P1C1).

    Techniques Used: Western Blot, Immunostaining

    10) Product Images from "Effect of troglitazone on plasma lipid metabolism and lipoprotein lipase"

    Article Title: Effect of troglitazone on plasma lipid metabolism and lipoprotein lipase

    Journal: British Journal of Clinical Pharmacology

    doi: 10.1046/j.1365-2125.1999.00920.x

    Lipoprotein lipase mass in culture media from 3T3 L1 cells cultured with or without troglitazone. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. After confluence, the cells were treated with differentiation medium (basal medium supplemented with 5% FBS,1 mg of insulin, 1 μm dexamethasone) with or without 10, 20 or 30 μm troglitazone for 6–7 days, replacing the media with fresh differentiation medium every 3 days. After the culture, media were harvested for measurement of lipoprotein lipase (LPL). Troglitazone was added to the media by dissolving the drug in dimethyl sulphoxide (DMSO) and diluting the drug 1000 times with media. LPL mass in cultured media from 3T3 cells was measured using an enzyme linked immunoassay as previously reported [11]. ***P
    Figure Legend Snippet: Lipoprotein lipase mass in culture media from 3T3 L1 cells cultured with or without troglitazone. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. After confluence, the cells were treated with differentiation medium (basal medium supplemented with 5% FBS,1 mg of insulin, 1 μm dexamethasone) with or without 10, 20 or 30 μm troglitazone for 6–7 days, replacing the media with fresh differentiation medium every 3 days. After the culture, media were harvested for measurement of lipoprotein lipase (LPL). Troglitazone was added to the media by dissolving the drug in dimethyl sulphoxide (DMSO) and diluting the drug 1000 times with media. LPL mass in cultured media from 3T3 cells was measured using an enzyme linked immunoassay as previously reported [11]. ***P

    Techniques Used: Cell Culture, Modification

    Northern blot analysis of RNA in 3T3-L1 cells. Total RNA was isolated from the 3T3-L1 cells cultured in the presence (left two lanes) or absence of (right two lanes) 10 μm troglitazone, and 20 μg of RNA samples were examined by Northern blot analysis. Hybridization was carried out over night in the presence of 32 P-labelled carboxyl terminal of mouse LPL (405 bp) cDNA.
    Figure Legend Snippet: Northern blot analysis of RNA in 3T3-L1 cells. Total RNA was isolated from the 3T3-L1 cells cultured in the presence (left two lanes) or absence of (right two lanes) 10 μm troglitazone, and 20 μg of RNA samples were examined by Northern blot analysis. Hybridization was carried out over night in the presence of 32 P-labelled carboxyl terminal of mouse LPL (405 bp) cDNA.

    Techniques Used: Northern Blot, Isolation, Cell Culture, Hybridization

    11) Product Images from "Sirt2 Regulates Adipocyte Differentiation Involving FoxO1 Acetylation/Deacetylation"

    Article Title: Sirt2 Regulates Adipocyte Differentiation Involving FoxO1 Acetylation/Deacetylation

    Journal:

    doi: 10.1016/j.cmet.2007.07.003

    Sirt2 knockdown promotes 3T3L1 adipocyte differentiation. Stable shRNA transfected 3T3-L1 preadipocytes were subjected to differentiation using the standard protocol. Oil Red O staining of shGFP and shSirt2 cells on day 4 of differentiation indicated
    Figure Legend Snippet: Sirt2 knockdown promotes 3T3L1 adipocyte differentiation. Stable shRNA transfected 3T3-L1 preadipocytes were subjected to differentiation using the standard protocol. Oil Red O staining of shGFP and shSirt2 cells on day 4 of differentiation indicated

    Techniques Used: shRNA, Transfection, Staining

    Sirt2 overexpression inhibits 3T3-L1 adipocyte differentiation without affecting insulin signaling in preadipocytes. (A). Following the differentiation protocol described in Material and Methods, Oil Red O staining of stably transfected 3T3-L1 cells with
    Figure Legend Snippet: Sirt2 overexpression inhibits 3T3-L1 adipocyte differentiation without affecting insulin signaling in preadipocytes. (A). Following the differentiation protocol described in Material and Methods, Oil Red O staining of stably transfected 3T3-L1 cells with

    Techniques Used: Over Expression, Staining, Stable Transfection, Transfection

    FoxO1 acetylation/deacetylation mimics regulate 3T3-L1 adipocyte differentiation and FoxO1 phosphorylation. (A) Different FoxO1 overexpression constructs were made with either wild type FoxO1 amino acid sequence or replacing all three lysine residues
    Figure Legend Snippet: FoxO1 acetylation/deacetylation mimics regulate 3T3-L1 adipocyte differentiation and FoxO1 phosphorylation. (A) Different FoxO1 overexpression constructs were made with either wild type FoxO1 amino acid sequence or replacing all three lysine residues

    Techniques Used: Over Expression, Construct, Sequencing

    12) Product Images from "Wake-sleep cycles are severely disrupted by diseases affecting cytoplasmic homeostasis"

    Article Title: Wake-sleep cycles are severely disrupted by diseases affecting cytoplasmic homeostasis

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

    doi: 10.1073/pnas.2003524117

    Spatial regulation of PER is directly linked to temporal manifestation of PER and circadian rhythms. ( A and B ) Fat vacuoles (Oil Red O) were enriched in the cytoplasm of adipocytes differentiated from 3T3 L1 cells. A zoomed-in image from the right image in A is shown in B . ( C ) Both hypophosphorylated and hyperphosphorylated PER species were visible at all times in the adipocytes. Unsynchronized cells are shown in the last two lanes. ( D ) Circadian rhythms were gradually compromised as L1 cells were differentiated into adipocytes and completely lost on day 8. Note that circadian period is unstable and generally longer in partially differentiated day 4 and day 6 cells compared with controls. The periods were estimated by peak-to-peak (red arrows) analysis in the samples. Three different symbols represent three independent samples. Data are mean ± SD, representative of two experiments. ( E ) Our model predicts that arrhythmicity in adipocytes can be rescued by increasing Per promoter activity. ( F ) Robust bioluminescence rhythms were recovered in adipocytes when PER2 was overexpressed using an adenoviral vector-delivered Per2 transgene ( Per2 promoter and Per2 coding sequence). n = 3 each. Data are representative of two experiments.
    Figure Legend Snippet: Spatial regulation of PER is directly linked to temporal manifestation of PER and circadian rhythms. ( A and B ) Fat vacuoles (Oil Red O) were enriched in the cytoplasm of adipocytes differentiated from 3T3 L1 cells. A zoomed-in image from the right image in A is shown in B . ( C ) Both hypophosphorylated and hyperphosphorylated PER species were visible at all times in the adipocytes. Unsynchronized cells are shown in the last two lanes. ( D ) Circadian rhythms were gradually compromised as L1 cells were differentiated into adipocytes and completely lost on day 8. Note that circadian period is unstable and generally longer in partially differentiated day 4 and day 6 cells compared with controls. The periods were estimated by peak-to-peak (red arrows) analysis in the samples. Three different symbols represent three independent samples. Data are mean ± SD, representative of two experiments. ( E ) Our model predicts that arrhythmicity in adipocytes can be rescued by increasing Per promoter activity. ( F ) Robust bioluminescence rhythms were recovered in adipocytes when PER2 was overexpressed using an adenoviral vector-delivered Per2 transgene ( Per2 promoter and Per2 coding sequence). n = 3 each. Data are representative of two experiments.

    Techniques Used: Activity Assay, Plasmid Preparation, Sequencing

    13) Product Images from "Monomeric Tartrate Resistant Acid Phosphatase Induces Insulin Sensitive Obesity"

    Article Title: Monomeric Tartrate Resistant Acid Phosphatase Induces Insulin Sensitive Obesity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001713

    Stimulation of adipocyte proliferation and differentiation by monomeric TRAP but not proteolytically processed TRAP. The following cells were used: Mouse 3T3-L1 preadipocytes (A, B), Human mesenchymal stem cells (hMSC) (C, D) and human pre-adipocytes (E, F). Proliferation (BrdU) was assessed after 1 day (3T3-L1), 2 days (3T3-L1, hMSC), 3 days (3T3-L1) or 5 days (pre-adipocytes) after seeding. Differentiation (GPDH-activity) was measured 4 days (3T3-L1) or 12 days (hMSC, pre-adipocytes) after start of differentiation. hMSC were treated with TRAP either before (Pro) or at (Post) confluence. mTRAP = monomeric TRAP, cTRAP = proteolytically processed TRAP. Data are expressed as percentage of control. Statistical analysis was performed using T-test, * p
    Figure Legend Snippet: Stimulation of adipocyte proliferation and differentiation by monomeric TRAP but not proteolytically processed TRAP. The following cells were used: Mouse 3T3-L1 preadipocytes (A, B), Human mesenchymal stem cells (hMSC) (C, D) and human pre-adipocytes (E, F). Proliferation (BrdU) was assessed after 1 day (3T3-L1), 2 days (3T3-L1, hMSC), 3 days (3T3-L1) or 5 days (pre-adipocytes) after seeding. Differentiation (GPDH-activity) was measured 4 days (3T3-L1) or 12 days (hMSC, pre-adipocytes) after start of differentiation. hMSC were treated with TRAP either before (Pro) or at (Post) confluence. mTRAP = monomeric TRAP, cTRAP = proteolytically processed TRAP. Data are expressed as percentage of control. Statistical analysis was performed using T-test, * p

    Techniques Used: Activity Assay

    14) Product Images from "Thrap3 docks on phosphoserine 273 of PPARγ and controls diabetic gene programming"

    Article Title: Thrap3 docks on phosphoserine 273 of PPARγ and controls diabetic gene programming

    Journal: Genes & Development

    doi: 10.1101/gad.249367.114

    Thrap3 specifically controls a diabetic gene set in cultured adipocytes. ( A ) 3T3-L1 cells expressing Scr or Thrap3 shRNA were stained with Oil-Red-O 7 d after adipocyte differentiation. ( B ) Expression of Thrap3 protein was analyzed with anti-Thrap3 antibody. ( C ) Specific genes that are dysregulated by CDK5/PPARγ phosphorylation expression were analyzed by real-time quantitative PCR (qPCR) ( n = 3). Error bars are SEM. (*) P
    Figure Legend Snippet: Thrap3 specifically controls a diabetic gene set in cultured adipocytes. ( A ) 3T3-L1 cells expressing Scr or Thrap3 shRNA were stained with Oil-Red-O 7 d after adipocyte differentiation. ( B ) Expression of Thrap3 protein was analyzed with anti-Thrap3 antibody. ( C ) Specific genes that are dysregulated by CDK5/PPARγ phosphorylation expression were analyzed by real-time quantitative PCR (qPCR) ( n = 3). Error bars are SEM. (*) P

    Techniques Used: Cell Culture, Expressing, shRNA, Staining, Real-time Polymerase Chain Reaction

    15) Product Images from "Eurycomanone regulates lipid metabolism by activating the cAMP/PKA pathway"

    Article Title: Eurycomanone regulates lipid metabolism by activating the cAMP/PKA pathway

    Journal: bioRxiv

    doi: 10.1101/2020.08.20.258855

    3T3-L1 pre-adipocytcs were treated with 25, 50,100 and 200 EN 72 h. (A) 3T3-L1 adipocytes were treated with 50,100 and 200 EN 24 h (B). The level in control cells was regarded as 1. Cell viability was assessed by MTT assay. The level in control cells was regarded as 100%. Values arc means ± SD (n=4). According to the student’s t test, there was no significant difference between the dosing group and the control cells.
    Figure Legend Snippet: 3T3-L1 pre-adipocytcs were treated with 25, 50,100 and 200 EN 72 h. (A) 3T3-L1 adipocytes were treated with 50,100 and 200 EN 24 h (B). The level in control cells was regarded as 1. Cell viability was assessed by MTT assay. The level in control cells was regarded as 100%. Values arc means ± SD (n=4). According to the student’s t test, there was no significant difference between the dosing group and the control cells.

    Techniques Used: MTT Assay

    Silence PKA-CA Gene pair 3T3-L1 Effect of lipolysis. (A) shows the results of glycerol release after treatment of EN (30 μM), siRNA, EN (30 μM) + siRNA on adipocytes, (B) indicates EN (30 μM), siRNA The effect of protein levels on C-AMP, P-PKA and P-HSL (C) indicates that the treated after 24 hours of treatment of cells, the effect of C-, EN (30 μM) + siRNA AMP, PKACA, PKA-CB, AKT and HSL mRNA expression levels. The level in ∞ntrol cells was set at 1. Values arc means ± SD (n=3), Data were analyzed by using Student’s t-test. *p
    Figure Legend Snippet: Silence PKA-CA Gene pair 3T3-L1 Effect of lipolysis. (A) shows the results of glycerol release after treatment of EN (30 μM), siRNA, EN (30 μM) + siRNA on adipocytes, (B) indicates EN (30 μM), siRNA The effect of protein levels on C-AMP, P-PKA and P-HSL (C) indicates that the treated after 24 hours of treatment of cells, the effect of C-, EN (30 μM) + siRNA AMP, PKACA, PKA-CB, AKT and HSL mRNA expression levels. The level in ∞ntrol cells was set at 1. Values arc means ± SD (n=3), Data were analyzed by using Student’s t-test. *p

    Techniques Used: Expressing

    3T3-L1 adipocytes were pretreated with 30 μM EN for 24 h. Protein expression was determined by Western blot assay. (E-F) compared to the positive ∞ntrol group, the effect of EN on the protein levels of P-PKA and P-HSL after 24 hours of treatment, and the gray value, (G) The effect of EN on the mRNA expression levels of C-AMP, ATGL, PKA-CA, PKA-CB, AKT and HSL after 24 hours of treatment with positive ∞ntrol group, (H-I) indicates the effect of EN after the action of PKA inhibitor H89 And the effect of ISO on the protein levels of CAMP, P-PKA and P-HSL after 24 hours of treatment of cells and the gray value. Data were analyzed by using Student’s t-test. *p
    Figure Legend Snippet: 3T3-L1 adipocytes were pretreated with 30 μM EN for 24 h. Protein expression was determined by Western blot assay. (E-F) compared to the positive ∞ntrol group, the effect of EN on the protein levels of P-PKA and P-HSL after 24 hours of treatment, and the gray value, (G) The effect of EN on the mRNA expression levels of C-AMP, ATGL, PKA-CA, PKA-CB, AKT and HSL after 24 hours of treatment with positive ∞ntrol group, (H-I) indicates the effect of EN after the action of PKA inhibitor H89 And the effect of ISO on the protein levels of CAMP, P-PKA and P-HSL after 24 hours of treatment of cells and the gray value. Data were analyzed by using Student’s t-test. *p

    Techniques Used: Expressing, Western Blot

    (A) shows the results of adding EN (30 μM) at four different time periods of 0-2 days, 2-4 days, 4-6 days, 6-B days. (B) Accumulation of intracellular triglycerides after addition of 3T?1 prc-adipocytes with EN at different time points. (C) Cell differentiation morphology of 3T3-L1 prcadipocytcs at 15 μM, 30 μM, 45 μM and 2-4 days after treatment with EN. (D) Accumulation of intracellular triglycerides after 3T-?1 prcadipocytcs were treated with EN. (E) Cell differentiation morphology of adipocytes under laser confocal microscopy at !5μM, 30 μM, and 45μM. (F) Results of glycerol release under the action of three concentrations of EN at 15 μM, 30 μM, and 45 μM. Values arc means ± SO (n=3). Data were analyzed by using Student’s t-test. *p
    Figure Legend Snippet: (A) shows the results of adding EN (30 μM) at four different time periods of 0-2 days, 2-4 days, 4-6 days, 6-B days. (B) Accumulation of intracellular triglycerides after addition of 3T?1 prc-adipocytes with EN at different time points. (C) Cell differentiation morphology of 3T3-L1 prcadipocytcs at 15 μM, 30 μM, 45 μM and 2-4 days after treatment with EN. (D) Accumulation of intracellular triglycerides after 3T-?1 prcadipocytcs were treated with EN. (E) Cell differentiation morphology of adipocytes under laser confocal microscopy at !5μM, 30 μM, and 45μM. (F) Results of glycerol release under the action of three concentrations of EN at 15 μM, 30 μM, and 45 μM. Values arc means ± SO (n=3). Data were analyzed by using Student’s t-test. *p

    Techniques Used: Cell Differentiation, Confocal Microscopy

    3T3-L1 adipocytes were pretreated with 30 μM EN for 24 h. Protein expression was determined by Western blot assay. One representative blot from three independent experiments is shown. The protein levels of the bands were quantified by densitometry. The effect of protein levels on C-AMP, P-PKA and P-HSL and the gray value after 24 hours of treatment of cells, (C-D) indicates that EN treated C-AMP, ATGL, PKA-CA, PKA-after 24 hours of treatment of cells. The effect of PKA-CB, AKT and HSL mRNA expression levels. (EF) compared to the positive control group, the effect of EN on the protein levels of P-PKA and P-HSL after 24 hours of treatment, and the gray value, (G) The effect of EN on the mRNA expression levels of C-AMP, ATGL, PKA-CA, PKA-CB, AKT and HSL after 24 hours of treatment with positive control group, (H-I) indicates the effect of EN after the action of PKA inhibitor H89 And the effect of ISO on the protein levels of C-AMP, P-PKA and P-HSL after 24 hours of treatment of cells and the gray value. (J) P-HSL protein expression under laser confocal scanning microscopy. The level in control cells was set at 1. Values arc means ± SD (n=3), Data were analyzed by using Student’s t-test. *p
    Figure Legend Snippet: 3T3-L1 adipocytes were pretreated with 30 μM EN for 24 h. Protein expression was determined by Western blot assay. One representative blot from three independent experiments is shown. The protein levels of the bands were quantified by densitometry. The effect of protein levels on C-AMP, P-PKA and P-HSL and the gray value after 24 hours of treatment of cells, (C-D) indicates that EN treated C-AMP, ATGL, PKA-CA, PKA-after 24 hours of treatment of cells. The effect of PKA-CB, AKT and HSL mRNA expression levels. (EF) compared to the positive control group, the effect of EN on the protein levels of P-PKA and P-HSL after 24 hours of treatment, and the gray value, (G) The effect of EN on the mRNA expression levels of C-AMP, ATGL, PKA-CA, PKA-CB, AKT and HSL after 24 hours of treatment with positive control group, (H-I) indicates the effect of EN after the action of PKA inhibitor H89 And the effect of ISO on the protein levels of C-AMP, P-PKA and P-HSL after 24 hours of treatment of cells and the gray value. (J) P-HSL protein expression under laser confocal scanning microscopy. The level in control cells was set at 1. Values arc means ± SD (n=3), Data were analyzed by using Student’s t-test. *p

    Techniques Used: Expressing, Western Blot, Positive Control, Confocal Laser Scanning Microscopy

    3T3-L1 adipocytes were pretreated with 30 μM EN for 24 h. Protein expression was determined by Western blot assay. One representative blot from three independent experiments is shown. The protein levels of the bands were quantified by densitometry. The effect of P-AKT, P-PKA and P-HSL on protein levels and gray-scale W values after 24 hours of treatment of cells, (E) indicates EN and MK versus P-AKT, P-PKA and P-HSL after 24 hours of treatment of adipocytes, Results of glycerol release under the action of concentrations of EN at 30 μM, MK. Values arc means ± SD (n=3). Data were analyzed by using Student’s t-test. * p
    Figure Legend Snippet: 3T3-L1 adipocytes were pretreated with 30 μM EN for 24 h. Protein expression was determined by Western blot assay. One representative blot from three independent experiments is shown. The protein levels of the bands were quantified by densitometry. The effect of P-AKT, P-PKA and P-HSL on protein levels and gray-scale W values after 24 hours of treatment of cells, (E) indicates EN and MK versus P-AKT, P-PKA and P-HSL after 24 hours of treatment of adipocytes, Results of glycerol release under the action of concentrations of EN at 30 μM, MK. Values arc means ± SD (n=3). Data were analyzed by using Student’s t-test. * p

    Techniques Used: Expressing, Western Blot

    16) Product Images from "The clock gene, brain and muscle Arnt-like 1, regulates adipogenesis via Wnt signaling pathway"

    Article Title: The clock gene, brain and muscle Arnt-like 1, regulates adipogenesis via Wnt signaling pathway

    Journal: The FASEB Journal

    doi: 10.1096/fj.12-205781

    Stable Bmal1 KD promoted adipogenesis during early differentiation of 3T3-L1 preadipocytes. A ) mRNA level of Bmal1 and Rev-erbα in SC and KD (shBmal1) cells, as determined by quantitative RT-PCR ( n =3). B ) Western blot analysis of Bmal1 protein. C ) Oil-red-O staining at d 3 of differentiation. D ) Gene expression analysis during differentiation, as determined by RT-qPCR ( n =3). * P
    Figure Legend Snippet: Stable Bmal1 KD promoted adipogenesis during early differentiation of 3T3-L1 preadipocytes. A ) mRNA level of Bmal1 and Rev-erbα in SC and KD (shBmal1) cells, as determined by quantitative RT-PCR ( n =3). B ) Western blot analysis of Bmal1 protein. C ) Oil-red-O staining at d 3 of differentiation. D ) Gene expression analysis during differentiation, as determined by RT-qPCR ( n =3). * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Staining, Expressing

    17) Product Images from "The repertoire of Adhesion G protein-coupled receptors in adipocytes and their functional relevance"

    Article Title: The repertoire of Adhesion G protein-coupled receptors in adipocytes and their functional relevance

    Journal: International Journal of Obesity (2005)

    doi: 10.1038/s41366-020-0570-2

    Expression of aGPCR during adipogenesis of 3T3-L1 cells. mRNA levels were determined by qPCR at six time points in the 10-day period of adipocyte differentiation and normalized to the housekeeping gene β actin (Ct = 17.23 ± 0.1). ΔCt values at day 0 are noted in dashed line box. Values of each day were further computed as relative fold change over day 0 expression and expression patterns were constructed. Raw data of qPCR measurements are noted in Supplementary Table 5 . Given is the mean ± SEM ( n = 4 biological replicates).
    Figure Legend Snippet: Expression of aGPCR during adipogenesis of 3T3-L1 cells. mRNA levels were determined by qPCR at six time points in the 10-day period of adipocyte differentiation and normalized to the housekeeping gene β actin (Ct = 17.23 ± 0.1). ΔCt values at day 0 are noted in dashed line box. Values of each day were further computed as relative fold change over day 0 expression and expression patterns were constructed. Raw data of qPCR measurements are noted in Supplementary Table 5 . Given is the mean ± SEM ( n = 4 biological replicates).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Construct

    Activation of endogenous GPR64 in 3T3-L1 cells and primary adipocytes. a Activation of endogenous GPR64 in 3T3-L1 preadipocytes using 0.5 mM agonistic peptide increases intracellular cAMP. Receptor knockdown with siRNA specific for Gpr64 leads to a significantly reduced cAMP accumulation. A scrambled version of the Stachel-peptide (0.5 mM) does not change intracellular cAMP levels. Given is the mean ± SEM of three independent experiments each performed in triplicates (cAMP concentration: Gpr64 siRNA: 3.56 ± 0.65 nM; control siRNA: 2.83 ± 0.33 nM). b Stimulation of mature 3T3-L1 adipocytes with pGPR64 at given concentrations significantly decreases the amount of secreted adiponectin similar to the effect of isoprenaline. As expected, the scrambled peptide does not have an effect on adiponectin secretion. Depicted is the mean ± SEM of five to six independent experiments performed in duplicates. c Glucose uptake was measured in fully differentiated 3T3-L1 cells after incubation with insulin, 0.5 mM pGPR64, 0.5 mM scGPR64, and 1 µM isoprenaline. Insulin induces a significant increase in glucose uptake which is reduced by isoprenaline. The stimulating peptide pGPR64 shows a trend towards reduced insulin-induced glucose uptake, whereas the scrambled peptide does not have an effect. Given is the mean ± SEM of two (isoprenaline) to four (pGPR64, scGPR64) independent experiments. Basal glucose uptake was 2.14 ± 0.34 dpm/mg protein. d Stimulation of GPR64 with the agonistic peptide pGPR64 (0.5 mM) results in a significantly enhanced lipolysis in mature 3T3-L1 and primary adipocytes. As expected, control stimulation of endogenously expressed β adrenergic receptors with isoprenaline increases lipolysis, while the scrambled peptide (at same concentration as the agonistic peptide) does not alter this function. Shown is the mean ± SEM of eight independent experiments performed in triplicates (3T3-L1, basal glycerol release 27.4 ± 2.36 µg/ml) or six independent experiments done in duplicates (primary adipocytes, basal glycerol release 17.4 ± 1.59 µg/ml). Statistical significance was tested using a paired t -test * p
    Figure Legend Snippet: Activation of endogenous GPR64 in 3T3-L1 cells and primary adipocytes. a Activation of endogenous GPR64 in 3T3-L1 preadipocytes using 0.5 mM agonistic peptide increases intracellular cAMP. Receptor knockdown with siRNA specific for Gpr64 leads to a significantly reduced cAMP accumulation. A scrambled version of the Stachel-peptide (0.5 mM) does not change intracellular cAMP levels. Given is the mean ± SEM of three independent experiments each performed in triplicates (cAMP concentration: Gpr64 siRNA: 3.56 ± 0.65 nM; control siRNA: 2.83 ± 0.33 nM). b Stimulation of mature 3T3-L1 adipocytes with pGPR64 at given concentrations significantly decreases the amount of secreted adiponectin similar to the effect of isoprenaline. As expected, the scrambled peptide does not have an effect on adiponectin secretion. Depicted is the mean ± SEM of five to six independent experiments performed in duplicates. c Glucose uptake was measured in fully differentiated 3T3-L1 cells after incubation with insulin, 0.5 mM pGPR64, 0.5 mM scGPR64, and 1 µM isoprenaline. Insulin induces a significant increase in glucose uptake which is reduced by isoprenaline. The stimulating peptide pGPR64 shows a trend towards reduced insulin-induced glucose uptake, whereas the scrambled peptide does not have an effect. Given is the mean ± SEM of two (isoprenaline) to four (pGPR64, scGPR64) independent experiments. Basal glucose uptake was 2.14 ± 0.34 dpm/mg protein. d Stimulation of GPR64 with the agonistic peptide pGPR64 (0.5 mM) results in a significantly enhanced lipolysis in mature 3T3-L1 and primary adipocytes. As expected, control stimulation of endogenously expressed β adrenergic receptors with isoprenaline increases lipolysis, while the scrambled peptide (at same concentration as the agonistic peptide) does not alter this function. Shown is the mean ± SEM of eight independent experiments performed in triplicates (3T3-L1, basal glycerol release 27.4 ± 2.36 µg/ml) or six independent experiments done in duplicates (primary adipocytes, basal glycerol release 17.4 ± 1.59 µg/ml). Statistical significance was tested using a paired t -test * p

    Techniques Used: Activation Assay, Concentration Assay, Incubation

    Effects of aGPCR knockdown on adipogenesis. 3T3-L1 cells were induced to differentiate under transient knockdown of the given aGPCR and compared with control-transfected cells. a , b , c Significant regulation of the adipogenic marker PPARγ under individual knockdown of six receptors was observed during adipogenesis. During the differentiation, we detected three different patterns of PPARγ expression (see Text for details). d Total lipid accumulation was measured by eluted ORO in day 10 adipocytes under receptor-specific transient knockdown and compared with control-transfected cells (Supplementary Table S7 ). e The count of lipid droplets per field of view (0.2664 mm 2 , minimum 5000 droplets counted per experiment) was lowered under knockdown of four receptors compared with control siRNA-transfected cells (Supplementary Table S7 ). f Lipid droplet size was significantly smaller under knockdown of Gpr124 and Gpr126 compared with control siRNA-transfected cells (Supplementary Table S7 ). g , h , i Analysis of lipid droplet size distribution. Size distribution of control is depicted in white bars (min to max). Given is the mean ± SEM ( n > 3 biological replicates). Statistical significance of PPARγ expression, ORO elution and lipid droplet size and count was identified by paired t -test. Lipid droplet size distribution was tested by two-way ANOVA followed by Dunnett’s test for multiple comparisons. * p
    Figure Legend Snippet: Effects of aGPCR knockdown on adipogenesis. 3T3-L1 cells were induced to differentiate under transient knockdown of the given aGPCR and compared with control-transfected cells. a , b , c Significant regulation of the adipogenic marker PPARγ under individual knockdown of six receptors was observed during adipogenesis. During the differentiation, we detected three different patterns of PPARγ expression (see Text for details). d Total lipid accumulation was measured by eluted ORO in day 10 adipocytes under receptor-specific transient knockdown and compared with control-transfected cells (Supplementary Table S7 ). e The count of lipid droplets per field of view (0.2664 mm 2 , minimum 5000 droplets counted per experiment) was lowered under knockdown of four receptors compared with control siRNA-transfected cells (Supplementary Table S7 ). f Lipid droplet size was significantly smaller under knockdown of Gpr124 and Gpr126 compared with control siRNA-transfected cells (Supplementary Table S7 ). g , h , i Analysis of lipid droplet size distribution. Size distribution of control is depicted in white bars (min to max). Given is the mean ± SEM ( n > 3 biological replicates). Statistical significance of PPARγ expression, ORO elution and lipid droplet size and count was identified by paired t -test. Lipid droplet size distribution was tested by two-way ANOVA followed by Dunnett’s test for multiple comparisons. * p

    Techniques Used: Transfection, Marker, Expressing

    18) Product Images from "Obesity-associated Family with Sequence Similarity 13, Member A (FAM13A) is Dispensable for Adipose Development and Insulin Sensitivity"

    Article Title: Obesity-associated Family with Sequence Similarity 13, Member A (FAM13A) is Dispensable for Adipose Development and Insulin Sensitivity

    Journal: International journal of obesity (2005)

    doi: 10.1038/s41366-018-0222-y

    Fam13a is upregulated during adipocyte differentiation potentially through PPARγ. a-b Time course of Fam13a ( a ) mRNA and ( b ) protein expression during induced differentiation of 3T3-L1 cells by DMI. The expression of Ap2 mRNA and PPARγ protein was used as positive controls. c Time course of Fam13a and Plin1 mRNA expression during induced differentiation of mouse primary stromal vascular cells (SVCs) by DMI. **: p
    Figure Legend Snippet: Fam13a is upregulated during adipocyte differentiation potentially through PPARγ. a-b Time course of Fam13a ( a ) mRNA and ( b ) protein expression during induced differentiation of 3T3-L1 cells by DMI. The expression of Ap2 mRNA and PPARγ protein was used as positive controls. c Time course of Fam13a and Plin1 mRNA expression during induced differentiation of mouse primary stromal vascular cells (SVCs) by DMI. **: p

    Techniques Used: Expressing

    FAM13A is nutritionally regulated and downregulated in genetic and diet-induced obesity. a-b Gene (n=6/group) and protein expression (n=3/group) of FAM13A in epididymal white adipose tissue (eWAT) of 3 months old male genetic obese ( a ) Lep ob/ob and ( b ) Lepr db/db mice as compared to their lean countparts. c-d Gene (n=10/group) and protein (n=3/group) expression of Fam13a in ( c ) eWAT and ( d ) Inguinal subcutaneous WAT (sWAT) of low fat diet (LFD) and high fat diet (HFD) fed male C57BL/6J mice. e-f mRNA expression of Fam13a in eWAT of 10 weeks old male C57BL/6J mice after ( e ) a 18 h fast and 6 h refeed; ( f ) 4 h fast (4hF) as compared to 48 h prolonged fasting (48hF) (n=7/group). g mRNA expression of Fam13a in mature 3T3-L1 adipocytes after 18 h treatment with dexamethasone (Dex) and insulin (Ins) as compared to vehicle-treated control (Ctrl) cells. Representative data were shown from 3 independent experiments in triplicates. **: p
    Figure Legend Snippet: FAM13A is nutritionally regulated and downregulated in genetic and diet-induced obesity. a-b Gene (n=6/group) and protein expression (n=3/group) of FAM13A in epididymal white adipose tissue (eWAT) of 3 months old male genetic obese ( a ) Lep ob/ob and ( b ) Lepr db/db mice as compared to their lean countparts. c-d Gene (n=10/group) and protein (n=3/group) expression of Fam13a in ( c ) eWAT and ( d ) Inguinal subcutaneous WAT (sWAT) of low fat diet (LFD) and high fat diet (HFD) fed male C57BL/6J mice. e-f mRNA expression of Fam13a in eWAT of 10 weeks old male C57BL/6J mice after ( e ) a 18 h fast and 6 h refeed; ( f ) 4 h fast (4hF) as compared to 48 h prolonged fasting (48hF) (n=7/group). g mRNA expression of Fam13a in mature 3T3-L1 adipocytes after 18 h treatment with dexamethasone (Dex) and insulin (Ins) as compared to vehicle-treated control (Ctrl) cells. Representative data were shown from 3 independent experiments in triplicates. **: p

    Techniques Used: Expressing, Mouse Assay

    Overexpression of murine FAM13A impairs preadipocyte survival. a FAM13A immunofluorescent staining, scale bar, 100 μm. b western blot and c quantification of β-Catenin expression; d qPCR analysis; e MTT viability assay; f Edu incorporation in vector (V) and FAM13A-overexpressing lentivirus transduced 3T3-L1 preadipocytes. g The levels of apoptosis evaluated by FACS analysis following APO-BrdU TUNEL staining combined with LIVE/DEAD cell staining in vector (V) and FAM13A-overexpressing lentivirus transduced 3T3-L1 preadipocytes with or without 24 h serum starvation. The picture shows one of the three experiments. h Numerical results of early and late apoptotic cells from FACS analysis. Values are presented as mean±SEM in 3 separate experiments *: p
    Figure Legend Snippet: Overexpression of murine FAM13A impairs preadipocyte survival. a FAM13A immunofluorescent staining, scale bar, 100 μm. b western blot and c quantification of β-Catenin expression; d qPCR analysis; e MTT viability assay; f Edu incorporation in vector (V) and FAM13A-overexpressing lentivirus transduced 3T3-L1 preadipocytes. g The levels of apoptosis evaluated by FACS analysis following APO-BrdU TUNEL staining combined with LIVE/DEAD cell staining in vector (V) and FAM13A-overexpressing lentivirus transduced 3T3-L1 preadipocytes with or without 24 h serum starvation. The picture shows one of the three experiments. h Numerical results of early and late apoptotic cells from FACS analysis. Values are presented as mean±SEM in 3 separate experiments *: p

    Techniques Used: Over Expression, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction, MTT Assay, Viability Assay, Plasmid Preparation, FACS, TUNEL Assay

    19) Product Images from "Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice"

    Article Title: Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice

    Journal: Nature Communications

    doi: 10.1038/ncomms14859

    Atglistatin transiently inhibits lipolysis and protects from HFD-induced obesity. Six weeks old male C57Bl6J mice were fed a HFD (45 kJ% fat; 22.1 kJ g −1 ) for 50 days. Thereafter, mice were fasted for 7 h and then re-fed a HFD with or without Atglistatin (2 mmol kg −1 diet) for 2 h followed by a second, subsequent fasting period of 8 h. ( a ) Plasma FA levels were determined in the 2 h re-fed and 8 h fasted state ( n =5). ( b ) FA release from gonadal adipose tissue explants of re-fed and ( c ) 8 h-fasted mice ( n =5 per group). ( d , e ) Atglistatin does not inhibit human adipocyte lipolysis. ( d ) TG hydrolase activity was assessed in COS-7 lysates of cells overexpressing human and murine ATGL and CGI-58, respectively, in the presence and absence of the indicated concentrations of Atglistatin. ( e ) SGBS and 3T3-L1 preadipocytes were differentiated to adipocytes. Then, cells were preincubated with the indicated concentrations of Atglistatin for 2 h. Thereafter, the medium was replaced by DMEM containing 2% BSA, 10 μM Forskolin and the indicated concentrations of Atglistatin for 1 h. The release of FA in the medium was determined and calculated per mg cell protein. ( f – k ) Mice were fed a HFD for 50 days, followed by HFD-feeding in the presence or absence of Atglistatin for another 50 days. ( f ) Body weight, and ( g ) fat and lean mass development ( n =7 per group). Adipose tissue depots, inguinal (i)WAT, gonadal (g)WAT, and interscapular (i)BAT were analysed for their ( h ) weight and ( i ) adipocyte size. ( j , k ) mRNA expression of IL-6 and macrophage markers F4/80 and Cd11c was assessed in gWAT and iBAT of re-fed mice ( n =5 per group). Data represent mean±s.d. Statistical significance was determined by Student's two-tailed t -test. For analysis of multiple measurements, we performed one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; * P
    Figure Legend Snippet: Atglistatin transiently inhibits lipolysis and protects from HFD-induced obesity. Six weeks old male C57Bl6J mice were fed a HFD (45 kJ% fat; 22.1 kJ g −1 ) for 50 days. Thereafter, mice were fasted for 7 h and then re-fed a HFD with or without Atglistatin (2 mmol kg −1 diet) for 2 h followed by a second, subsequent fasting period of 8 h. ( a ) Plasma FA levels were determined in the 2 h re-fed and 8 h fasted state ( n =5). ( b ) FA release from gonadal adipose tissue explants of re-fed and ( c ) 8 h-fasted mice ( n =5 per group). ( d , e ) Atglistatin does not inhibit human adipocyte lipolysis. ( d ) TG hydrolase activity was assessed in COS-7 lysates of cells overexpressing human and murine ATGL and CGI-58, respectively, in the presence and absence of the indicated concentrations of Atglistatin. ( e ) SGBS and 3T3-L1 preadipocytes were differentiated to adipocytes. Then, cells were preincubated with the indicated concentrations of Atglistatin for 2 h. Thereafter, the medium was replaced by DMEM containing 2% BSA, 10 μM Forskolin and the indicated concentrations of Atglistatin for 1 h. The release of FA in the medium was determined and calculated per mg cell protein. ( f – k ) Mice were fed a HFD for 50 days, followed by HFD-feeding in the presence or absence of Atglistatin for another 50 days. ( f ) Body weight, and ( g ) fat and lean mass development ( n =7 per group). Adipose tissue depots, inguinal (i)WAT, gonadal (g)WAT, and interscapular (i)BAT were analysed for their ( h ) weight and ( i ) adipocyte size. ( j , k ) mRNA expression of IL-6 and macrophage markers F4/80 and Cd11c was assessed in gWAT and iBAT of re-fed mice ( n =5 per group). Data represent mean±s.d. Statistical significance was determined by Student's two-tailed t -test. For analysis of multiple measurements, we performed one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; * P

    Techniques Used: Mouse Assay, Activity Assay, Expressing, Two Tailed Test

    20) Product Images from "Selective measurement of NAPE-PLD activity via a PLA1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog"

    Article Title: Selective measurement of NAPE-PLD activity via a PLA1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog

    Journal: Journal of Lipid Research

    doi: 10.1016/j.jlr.2021.100156

    Phospholipase activity measured using flame-NAPE is more sensitive to NAPE-PLD inhibition than activity measured using PED-A1 in many cell types. Various cultured cell types were treated with or without 15 μM bithionol (Bith), an NAPE-PLD inhibitor, prior to incubation with PED-A1 or flame-NAPE (4 μM). Bars represent average ± SEM, n = 31–48 with 7–16 replicates each for three separate experimental days. A: HEK-293, (B) 3T3-L1, (C) BMDM (bone marrow-derived macrophage), and (D) Caco-2. One-way ANOVA for each cell type P
    Figure Legend Snippet: Phospholipase activity measured using flame-NAPE is more sensitive to NAPE-PLD inhibition than activity measured using PED-A1 in many cell types. Various cultured cell types were treated with or without 15 μM bithionol (Bith), an NAPE-PLD inhibitor, prior to incubation with PED-A1 or flame-NAPE (4 μM). Bars represent average ± SEM, n = 31–48 with 7–16 replicates each for three separate experimental days. A: HEK-293, (B) 3T3-L1, (C) BMDM (bone marrow-derived macrophage), and (D) Caco-2. One-way ANOVA for each cell type P

    Techniques Used: Activity Assay, Inhibition, Cell Culture, Incubation, Derivative Assay

    21) Product Images from "Identification of DNA response elements regulating expression of CCAAT/enhancer-binding protein (C/EBP) β and δ during early adipogenesis"

    Article Title: Identification of DNA response elements regulating expression of CCAAT/enhancer-binding protein (C/EBP) β and δ during early adipogenesis

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.110114

    (A-C) 3T3-L1 fibroblasts were treated with differentiation medium for the indicated times (h or min, unless indicated by D = days) at which point cells were harvested and analysed by immunoblot using the indicated antibodies. Data are representative of at least three independent experiments.
    Figure Legend Snippet: (A-C) 3T3-L1 fibroblasts were treated with differentiation medium for the indicated times (h or min, unless indicated by D = days) at which point cells were harvested and analysed by immunoblot using the indicated antibodies. Data are representative of at least three independent experiments.

    Techniques Used:

    (A) Schematic diagram depicting the genomic position of putative response elements on th Mknk2 promoter relative to the TSS (chr10:80,139,038). The sequences of the putative response elements are displayed; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (B-D) PPARγ at putative PPAREs and (G) C/EBPα at putative C/EBPREs after 72 h of induction. (E,F) Enrichment of C/EBPβ at putative C/EBPREs was assessed after 24 h. (H-J) Enrichment was also assessed at the indicated NEG region. Data are mean ± SEM; n=2; unpaired two-tailed t-test (*P
    Figure Legend Snippet: (A) Schematic diagram depicting the genomic position of putative response elements on th Mknk2 promoter relative to the TSS (chr10:80,139,038). The sequences of the putative response elements are displayed; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (B-D) PPARγ at putative PPAREs and (G) C/EBPα at putative C/EBPREs after 72 h of induction. (E,F) Enrichment of C/EBPβ at putative C/EBPREs was assessed after 24 h. (H-J) Enrichment was also assessed at the indicated NEG region. Data are mean ± SEM; n=2; unpaired two-tailed t-test (*P

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Two Tailed Test

    (A,B) RT-qPCR analysis of Mknk1 and Mknk2 expression in the indicated tissues of C57BL6/J mice fed chow or a high fat diet for 16 weeks. Data presented are mean ± SEM (n=5-6 per group). (C,D) 3T3-L1 fibroblasts were treated with the differentiation medium for the indicated times (h, unless denoted by D = days) at which point samples were analysed for expression of Mknk1 and Mknk2 by RT-qPCR. (E,F) 3T3-L1 fibroblasts were treated with the indicated components of the differentiation medium or the entire cocktail, as indicated, for 3 h at which point samples were analysed for expression of Mknk1 and Mknk2 by RT-qPCR. Data presented are mean ± SEM (n=3).
    Figure Legend Snippet: (A,B) RT-qPCR analysis of Mknk1 and Mknk2 expression in the indicated tissues of C57BL6/J mice fed chow or a high fat diet for 16 weeks. Data presented are mean ± SEM (n=5-6 per group). (C,D) 3T3-L1 fibroblasts were treated with the differentiation medium for the indicated times (h, unless denoted by D = days) at which point samples were analysed for expression of Mknk1 and Mknk2 by RT-qPCR. (E,F) 3T3-L1 fibroblasts were treated with the indicated components of the differentiation medium or the entire cocktail, as indicated, for 3 h at which point samples were analysed for expression of Mknk1 and Mknk2 by RT-qPCR. Data presented are mean ± SEM (n=3).

    Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay

    (A) Schematic diagram depicting the genomic position of putative response elements on the Cebpb promoter relative to the TSS (chr2:167,514,466). The sequences of the putative response elements are displayed in the table; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (B,C) P-CREB at putative CREs and (D-F) GR at putative GREs after 3 h of adipogenic induction. (G,H) Enrichment was also assessed at the indicated NEG region. Data are mean ± SEM; n=4; unpaired two-tailed t-test (*P
    Figure Legend Snippet: (A) Schematic diagram depicting the genomic position of putative response elements on the Cebpb promoter relative to the TSS (chr2:167,514,466). The sequences of the putative response elements are displayed in the table; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (B,C) P-CREB at putative CREs and (D-F) GR at putative GREs after 3 h of adipogenic induction. (G,H) Enrichment was also assessed at the indicated NEG region. Data are mean ± SEM; n=4; unpaired two-tailed t-test (*P

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Two Tailed Test

    (A) Schematic diagram depicting the genomic position of putative response elements on the Cebpd promoter relative to the TSS (chr16:15,887,379). The sequences of the putative response elements are displayed in the table; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (B) P-CREB at putative CREs and (C) GR at putative GREs after 3 h of adipogenic induction. (D,E) Enrichment was also assessed at the indicated NEG region. Data are mean ± SEM; n=4; unpaired two-tailed t-test (*P
    Figure Legend Snippet: (A) Schematic diagram depicting the genomic position of putative response elements on the Cebpd promoter relative to the TSS (chr16:15,887,379). The sequences of the putative response elements are displayed in the table; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (B) P-CREB at putative CREs and (C) GR at putative GREs after 3 h of adipogenic induction. (D,E) Enrichment was also assessed at the indicated NEG region. Data are mean ± SEM; n=4; unpaired two-tailed t-test (*P

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Two Tailed Test

    (A-D) 3T3-L1 fibroblasts were treated with the differentiation medium for the indicated times (h, unless indicated by D = days) at which point samples were analysed for expression of Cebpb, Cebpd, Cebpa and Pparg by RT-qPCR. (E,F) 3T3-L1 fibroblasts were treated with the indicated components of the differentiation medium or the entire cocktail, as indicated, for 3 h at which point samples were analysed for expression of Cebpb and Cebpd by RT-qPCR. Data presented are mean ± SEM (n=3).
    Figure Legend Snippet: (A-D) 3T3-L1 fibroblasts were treated with the differentiation medium for the indicated times (h, unless indicated by D = days) at which point samples were analysed for expression of Cebpb, Cebpd, Cebpa and Pparg by RT-qPCR. (E,F) 3T3-L1 fibroblasts were treated with the indicated components of the differentiation medium or the entire cocktail, as indicated, for 3 h at which point samples were analysed for expression of Cebpb and Cebpd by RT-qPCR. Data presented are mean ± SEM (n=3).

    Techniques Used: Expressing, Quantitative RT-PCR

    Schematic diagram depicting the genomic position of putative response elements on the (A) Mknk1 and (F) Mknk2 promoters relative to their TSS (chr4:115,511,826 and chr10:80,139,038, respectively). The sequences of the putative response elements are displayed in the tables below; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (B,H) P-CREB at putative CREs and (C,G,I,J) GR at putative GREs after 3 h of adipogenic induction. (D,E,K,L) Enrichment was also assessed at the indicated NEG regions of the two genes. Data are mean ± SEM; n=4; unpaired two-tailed t-test (*P
    Figure Legend Snippet: Schematic diagram depicting the genomic position of putative response elements on the (A) Mknk1 and (F) Mknk2 promoters relative to their TSS (chr4:115,511,826 and chr10:80,139,038, respectively). The sequences of the putative response elements are displayed in the tables below; nucleotides corresponding to the core consensus sequence are shown bold and underlined. ChIP assays were performed in 3T3-L1 fibroblasts to assess enrichment of (B,H) P-CREB at putative CREs and (C,G,I,J) GR at putative GREs after 3 h of adipogenic induction. (D,E,K,L) Enrichment was also assessed at the indicated NEG regions of the two genes. Data are mean ± SEM; n=4; unpaired two-tailed t-test (*P

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Two Tailed Test

    22) Product Images from "Characterization of a proximal Sp1 response element in the mouse Dlk2 gene promoter"

    Article Title: Characterization of a proximal Sp1 response element in the mouse Dlk2 gene promoter

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-12-52

    Sp1 binds to the Dlk2 promoter . A) Schematic representation of the six GC-boxes in the Dlk2 promoter, the putative Sp1 binding sites (named 1, 2, 3, 4, 5, and 6). The oligonucleotides used for the ChIP analyses (arrows), and the approximate positions of three oligonucleotides used in EMSA assays are also shown. Oligonucleotide Sp1-A contains the GC boxes located at positions -130/-140 (GCBox-1); -108/-115 (GCBox-2); oligonucleotide Sp1-B contains the GC box located at position -61/-70 (GCBox-3); and oligonucleotide Sp1-C contains the GC boxes located at positions +62/+68 (GCBox-4); +76/+82 (GCBox-5); and +81/+90 (GCBox-6). B) Chromatin IP analysis was performed using native chromatin from 3T3-L1 cells, incubated with normal rabbit IgG (IgG), with antibodies against RNA-polymerase II (pol-II), or with antibodies against Sp1. The PCR analysis and the corresponding agarose gel electrophoresis of a representative experiment is shown. C) and D) EMSA analyses were performed using 8 μg of nuclear protein extracts from NIH3T3 cells, which were incubated with the 32 P-labeled oligonucleotides Sp1-A, Sp1-B or Sp1-C. For competition and super-shift assays, the reaction was preincubated with a 100-fold excess of the indicated cold oligonucleotides, or with 2 μg of Sp1 antibody before the addition of the labeled oligonucleotide. The locations of the Sp1 and Sp1 supershifted (SS) bands are indicated by arrows.
    Figure Legend Snippet: Sp1 binds to the Dlk2 promoter . A) Schematic representation of the six GC-boxes in the Dlk2 promoter, the putative Sp1 binding sites (named 1, 2, 3, 4, 5, and 6). The oligonucleotides used for the ChIP analyses (arrows), and the approximate positions of three oligonucleotides used in EMSA assays are also shown. Oligonucleotide Sp1-A contains the GC boxes located at positions -130/-140 (GCBox-1); -108/-115 (GCBox-2); oligonucleotide Sp1-B contains the GC box located at position -61/-70 (GCBox-3); and oligonucleotide Sp1-C contains the GC boxes located at positions +62/+68 (GCBox-4); +76/+82 (GCBox-5); and +81/+90 (GCBox-6). B) Chromatin IP analysis was performed using native chromatin from 3T3-L1 cells, incubated with normal rabbit IgG (IgG), with antibodies against RNA-polymerase II (pol-II), or with antibodies against Sp1. The PCR analysis and the corresponding agarose gel electrophoresis of a representative experiment is shown. C) and D) EMSA analyses were performed using 8 μg of nuclear protein extracts from NIH3T3 cells, which were incubated with the 32 P-labeled oligonucleotides Sp1-A, Sp1-B or Sp1-C. For competition and super-shift assays, the reaction was preincubated with a 100-fold excess of the indicated cold oligonucleotides, or with 2 μg of Sp1 antibody before the addition of the labeled oligonucleotide. The locations of the Sp1 and Sp1 supershifted (SS) bands are indicated by arrows.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Incubation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Labeling

    Characterization of the Dlk2 transcription start site . A) Schematic exon-intron distribution of Dlk2 cDNAs putatively assigned to Dlk2 . The arrows indicate the position of oligonucleotides used to specifically amplify Dlk2 transcripts. B) RT-PCR of Dlk2 transcripts with the following templates: cDNAs from 3T3-L1, NIH3T3, C3H10T1/2, or AT3F cells; cDNAs from heart (H), spleen (S), testicles (T), brain (B) and lung (L) of adult 129/C57BL6 mice; or genomic DNA (gDNA). Two PCR reactions were performed with each template, one to amplify variant 1 transcript (V1) and another to amplify variant 2 (V2). The sizes of the expected amplified DNA fragments are shown on the right. C) Analysis of Dlk2 mRNA expression levels in 3T3-L1, NIH3T3, C3H10T1/2 and AT3F cell lines by RT-qPCR. mRNA levels were referred to the expression level of phosphoriboprotein P0, which was used as an internal control. D) Experimental determination of Dlk2 TSS by 5' RACE. RNA from AT3F cells was used for 5' RACE amplification, using a specific reverse primer located within the sixth exon, at position +693 from the translation initiation codon (ATG), indicated by an arrow in the upper pannel. The amplified PCR products were cloned into the pCR2.1 vector, and twenty individual clones were sequenced. The 5' region sequences of genomic DNA and cDNA clone BC091431 are shown, including the 14 additional bases of the newly described clone FM180474.
    Figure Legend Snippet: Characterization of the Dlk2 transcription start site . A) Schematic exon-intron distribution of Dlk2 cDNAs putatively assigned to Dlk2 . The arrows indicate the position of oligonucleotides used to specifically amplify Dlk2 transcripts. B) RT-PCR of Dlk2 transcripts with the following templates: cDNAs from 3T3-L1, NIH3T3, C3H10T1/2, or AT3F cells; cDNAs from heart (H), spleen (S), testicles (T), brain (B) and lung (L) of adult 129/C57BL6 mice; or genomic DNA (gDNA). Two PCR reactions were performed with each template, one to amplify variant 1 transcript (V1) and another to amplify variant 2 (V2). The sizes of the expected amplified DNA fragments are shown on the right. C) Analysis of Dlk2 mRNA expression levels in 3T3-L1, NIH3T3, C3H10T1/2 and AT3F cell lines by RT-qPCR. mRNA levels were referred to the expression level of phosphoriboprotein P0, which was used as an internal control. D) Experimental determination of Dlk2 TSS by 5' RACE. RNA from AT3F cells was used for 5' RACE amplification, using a specific reverse primer located within the sixth exon, at position +693 from the translation initiation codon (ATG), indicated by an arrow in the upper pannel. The amplified PCR products were cloned into the pCR2.1 vector, and twenty individual clones were sequenced. The 5' region sequences of genomic DNA and cDNA clone BC091431 are shown, including the 14 additional bases of the newly described clone FM180474.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Polymerase Chain Reaction, Variant Assay, Amplification, Expressing, Quantitative RT-PCR, Clone Assay, Plasmid Preparation

    23) Product Images from "Betacyanins and Anthocyanins in Pulp and Peel of Red Pitaya (Hylocereus polyrhizus cv. Jindu), Inhibition of Oxidative Stress, Lipid Reducing, and Cytotoxic Effects"

    Article Title: Betacyanins and Anthocyanins in Pulp and Peel of Red Pitaya (Hylocereus polyrhizus cv. Jindu), Inhibition of Oxidative Stress, Lipid Reducing, and Cytotoxic Effects

    Journal: Frontiers in Nutrition

    doi: 10.3389/fnut.2022.894438

    (A) Antioxidant activity and (B) EC 50 values of H 2 O 2 -induced oxidative stress of 3T3-L1 cells. a Calculated based on the non-H 2 O 2 -induced control, where the oxidative stress was induced for 48 h. b IC 50 values were calculated based on the cell viability of 3T3-L1 cells. c Different lowercase letters indicate significant differences at p
    Figure Legend Snippet: (A) Antioxidant activity and (B) EC 50 values of H 2 O 2 -induced oxidative stress of 3T3-L1 cells. a Calculated based on the non-H 2 O 2 -induced control, where the oxidative stress was induced for 48 h. b IC 50 values were calculated based on the cell viability of 3T3-L1 cells. c Different lowercase letters indicate significant differences at p

    Techniques Used: Antioxidant Activity Assay

    Total antioxidant status (TAS) of H 2 O 2 -induced oxidative stress to 3T3-L1 cells. a TAS of 3T3-L1 cells induced by H 2 O 2 for 24 h. b Different lowercase letters indicate significant differences between two samples at p
    Figure Legend Snippet: Total antioxidant status (TAS) of H 2 O 2 -induced oxidative stress to 3T3-L1 cells. a TAS of 3T3-L1 cells induced by H 2 O 2 for 24 h. b Different lowercase letters indicate significant differences between two samples at p

    Techniques Used:

    Inhibition effect of lipid accumulation in 3T3-L1 cells treated with (A) pitaya samples and (B) standards. a Assessed based on the oil red O staining assay. b NDC, non-differentiated cells.
    Figure Legend Snippet: Inhibition effect of lipid accumulation in 3T3-L1 cells treated with (A) pitaya samples and (B) standards. a Assessed based on the oil red O staining assay. b NDC, non-differentiated cells.

    Techniques Used: Inhibition, Staining

    Percentages of cell viability of 3T3-L1 cells induced by H 2 O 2 for (A) 24 h and (B) 48 h.
    Figure Legend Snippet: Percentages of cell viability of 3T3-L1 cells induced by H 2 O 2 for (A) 24 h and (B) 48 h.

    Techniques Used:

    IC 50 values of pitaya samples and standards for cytotoxicity of (A) 3T3-L1 and (B) RIN-5F cell lines treated for 24 and 72 h. a Different lowercase letters indicate significant differences between different treatment times and between two samples at p
    Figure Legend Snippet: IC 50 values of pitaya samples and standards for cytotoxicity of (A) 3T3-L1 and (B) RIN-5F cell lines treated for 24 and 72 h. a Different lowercase letters indicate significant differences between different treatment times and between two samples at p

    Techniques Used:

    24) Product Images from "An optimized desuccinylase activity assay reveals a difference in desuccinylation activity between proliferative and differentiated cells"

    Article Title: An optimized desuccinylase activity assay reveals a difference in desuccinylation activity between proliferative and differentiated cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-72833-7

    NAD + -dependent deacylase activities in C2C12 and 3T3-L1 cell lysates. ( a ) Light micrograph of myoblasts (left panel), and myotubes at day 6 of differentiation (right panel); scale bar is 1000 µm. ( b ) Light micrograph of preadipocytes (left panel), and adipocytes at day 11 of differentiation (right panel); scale bar is 200 µm. NAD + -dependent desuccinylase activity in 150 µg lysates of ( c ) myoblast and myotubes and ( d ) 180 µg lysates of preadipocytes and adipocytes. NAD + -dependent deacetylase activity in lysates (145.6 µg) of ( e ) myoblasts and myotubes and ( f ) in lysates (145.6 µg) of preadipocytes and adipocytes. Date represent mean ± SEM. n = 3 for desuccinylation activity assays. Two-tailed unpaired Student’s t-test was performed. *P
    Figure Legend Snippet: NAD + -dependent deacylase activities in C2C12 and 3T3-L1 cell lysates. ( a ) Light micrograph of myoblasts (left panel), and myotubes at day 6 of differentiation (right panel); scale bar is 1000 µm. ( b ) Light micrograph of preadipocytes (left panel), and adipocytes at day 11 of differentiation (right panel); scale bar is 200 µm. NAD + -dependent desuccinylase activity in 150 µg lysates of ( c ) myoblast and myotubes and ( d ) 180 µg lysates of preadipocytes and adipocytes. NAD + -dependent deacetylase activity in lysates (145.6 µg) of ( e ) myoblasts and myotubes and ( f ) in lysates (145.6 µg) of preadipocytes and adipocytes. Date represent mean ± SEM. n = 3 for desuccinylation activity assays. Two-tailed unpaired Student’s t-test was performed. *P

    Techniques Used: Activity Assay, Histone Deacetylase Assay, Two Tailed Test

    SIRT5 protein and succinylation levels in C2C12 myoblasts and myotubes and in 3T3-L1 preadipocytes and adipocytes. ( a ) Western blot analysis of voltage-dependent anion channel (VDAC), SIRT5 and histone 3. The numbers represent band intensity relative to histone 3. Blots of VDAC and SIRT5 were cropped from the same gel, and histone 3 was cropped from a different gel. Full-length blots are presented in Supplementary Fig. 3 . ( b ) Succinyllysine levels and blot lane profiles of C2C12 myoblasts (black line) and myotubes (green line). Grey areas represent succinyllysine protein bands with higher intensity in myoblasts and green areas represent succinyllysine protein bands with lower intensity in myoblasts. ( c ) Succinyllysine levels and blot lane profiles of 3T3-L1 preadipocytes (black line) and adipocytes (red line). Grey areas represent succinyllysine protein bands with higher intensity in preadipocytes and red areas represent succinyllysine protein bands with lower intensity in adipocytes. In ( b , c ), blots of succinylation and histone3 were grouped from different gels, and full-length blots for ( b , c ) are presented in Supplementary Fig. 4 .
    Figure Legend Snippet: SIRT5 protein and succinylation levels in C2C12 myoblasts and myotubes and in 3T3-L1 preadipocytes and adipocytes. ( a ) Western blot analysis of voltage-dependent anion channel (VDAC), SIRT5 and histone 3. The numbers represent band intensity relative to histone 3. Blots of VDAC and SIRT5 were cropped from the same gel, and histone 3 was cropped from a different gel. Full-length blots are presented in Supplementary Fig. 3 . ( b ) Succinyllysine levels and blot lane profiles of C2C12 myoblasts (black line) and myotubes (green line). Grey areas represent succinyllysine protein bands with higher intensity in myoblasts and green areas represent succinyllysine protein bands with lower intensity in myoblasts. ( c ) Succinyllysine levels and blot lane profiles of 3T3-L1 preadipocytes (black line) and adipocytes (red line). Grey areas represent succinyllysine protein bands with higher intensity in preadipocytes and red areas represent succinyllysine protein bands with lower intensity in adipocytes. In ( b , c ), blots of succinylation and histone3 were grouped from different gels, and full-length blots for ( b , c ) are presented in Supplementary Fig. 4 .

    Techniques Used: Western Blot

    25) Product Images from "A Novel Non-agonist Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand UHC1 Blocks PPARγ Phosphorylation by Cyclin-dependent Kinase 5 (CDK5) and Improves Insulin Sensitivity *"

    Article Title: A Novel Non-agonist Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand UHC1 Blocks PPARγ Phosphorylation by Cyclin-dependent Kinase 5 (CDK5) and Improves Insulin Sensitivity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.566794

    Non-agonism of UHC1. a , transcriptional activity of a PPAR-derived reporter gene in HEK-293 cells following treatment with rosiglitazone ( Rosi ; 10 μmol/liter), UHC1 (10 μmol/liter), or SR1664 (10 μmol/liter). b , lipid accumulation in differentiated 3T3-L1 adipocytes following Oil-red O staining. Expression of adipocyte-enriched genes ( c ) and gene set regulated by PPARγ phosphorylation ( d ) in these cells was analyzed by qPCR. Error bars are S.E. ( n = 3). *, p
    Figure Legend Snippet: Non-agonism of UHC1. a , transcriptional activity of a PPAR-derived reporter gene in HEK-293 cells following treatment with rosiglitazone ( Rosi ; 10 μmol/liter), UHC1 (10 μmol/liter), or SR1664 (10 μmol/liter). b , lipid accumulation in differentiated 3T3-L1 adipocytes following Oil-red O staining. Expression of adipocyte-enriched genes ( c ) and gene set regulated by PPARγ phosphorylation ( d ) in these cells was analyzed by qPCR. Error bars are S.E. ( n = 3). *, p

    Techniques Used: Activity Assay, Derivative Assay, Staining, Expressing, Real-time Polymerase Chain Reaction

    Suppression of proinflammatory gene expression by UHC1 in vitro . a , differentiated 3T3-L1 adipocytes were incubated with rosiglitazone ( Rosi ), UHC1, or SR1664 for 24 h and were treated with TNF-α (10 ng/ml) for 3 h. b , Raw264.7 macrophages were incubated with rosiglitazone, UHC1, or SR1664 at specific concentration for 24 h and were treated with LPS (100 ng/ml) for 6 h. Relative gene expression was determined by qPCR. c , Raw264.7 cells were preincubated with UHC1 at specific concentration for 1 h and were treated with LPS (100 ng/ml) for 24 h. The amount of nitrite in cell-free culture supernatants was measured using Griess reagent. d , Raw264.7 macrophages expressing PPARγWT or PPARγS273A were treated with LPS (100 ng/ml) for 6 h. Relative gene expression was determined by qPCR. Error bars are S.E. ( n = 3). *, p
    Figure Legend Snippet: Suppression of proinflammatory gene expression by UHC1 in vitro . a , differentiated 3T3-L1 adipocytes were incubated with rosiglitazone ( Rosi ), UHC1, or SR1664 for 24 h and were treated with TNF-α (10 ng/ml) for 3 h. b , Raw264.7 macrophages were incubated with rosiglitazone, UHC1, or SR1664 at specific concentration for 24 h and were treated with LPS (100 ng/ml) for 6 h. Relative gene expression was determined by qPCR. c , Raw264.7 cells were preincubated with UHC1 at specific concentration for 1 h and were treated with LPS (100 ng/ml) for 24 h. The amount of nitrite in cell-free culture supernatants was measured using Griess reagent. d , Raw264.7 macrophages expressing PPARγWT or PPARγS273A were treated with LPS (100 ng/ml) for 6 h. Relative gene expression was determined by qPCR. Error bars are S.E. ( n = 3). *, p

    Techniques Used: Expressing, In Vitro, Incubation, Concentration Assay, Real-time Polymerase Chain Reaction

    26) Product Images from "Sirt2 Regulates Adipocyte Differentiation Involving FoxO1 Acetylation/Deacetylation"

    Article Title: Sirt2 Regulates Adipocyte Differentiation Involving FoxO1 Acetylation/Deacetylation

    Journal:

    doi: 10.1016/j.cmet.2007.07.003

    Sirt2 knockdown promotes 3T3L1 adipocyte differentiation. Stable shRNA transfected 3T3-L1 preadipocytes were subjected to differentiation using the standard protocol. Oil Red O staining of shGFP and shSirt2 cells on day 4 of differentiation indicated
    Figure Legend Snippet: Sirt2 knockdown promotes 3T3L1 adipocyte differentiation. Stable shRNA transfected 3T3-L1 preadipocytes were subjected to differentiation using the standard protocol. Oil Red O staining of shGFP and shSirt2 cells on day 4 of differentiation indicated

    Techniques Used: shRNA, Transfection, Staining

    Sirt2 overexpression inhibits 3T3-L1 adipocyte differentiation without affecting insulin signaling in preadipocytes. (A). Following the differentiation protocol described in Material and Methods, Oil Red O staining of stably transfected 3T3-L1 cells with
    Figure Legend Snippet: Sirt2 overexpression inhibits 3T3-L1 adipocyte differentiation without affecting insulin signaling in preadipocytes. (A). Following the differentiation protocol described in Material and Methods, Oil Red O staining of stably transfected 3T3-L1 cells with

    Techniques Used: Over Expression, Staining, Stable Transfection, Transfection

    FoxO1 acetylation/deacetylation mimics regulate 3T3-L1 adipocyte differentiation and FoxO1 phosphorylation. (A) Different FoxO1 overexpression constructs were made with either wild type FoxO1 amino acid sequence or replacing all three lysine residues
    Figure Legend Snippet: FoxO1 acetylation/deacetylation mimics regulate 3T3-L1 adipocyte differentiation and FoxO1 phosphorylation. (A) Different FoxO1 overexpression constructs were made with either wild type FoxO1 amino acid sequence or replacing all three lysine residues

    Techniques Used: Over Expression, Construct, Sequencing

    27) Product Images from "Inhibition of lipid accumulation by the ethyl acetate fraction of Distylium racemosum in vitro and in vivo"

    Article Title: Inhibition of lipid accumulation by the ethyl acetate fraction of Distylium racemosum in vitro and in vivo

    Journal: Toxicology Reports

    doi: 10.1016/j.toxrep.2019.02.003

    Effects of ethyl acetate fraction from Distylium racemosum on lipid accumulation in 3T3-L1 cells. Lipid accumulation was measured by Oil red O staining. All photographs were taken using a microscope. Lipid accumulation was inhibited by DRE (magnification ×400).
    Figure Legend Snippet: Effects of ethyl acetate fraction from Distylium racemosum on lipid accumulation in 3T3-L1 cells. Lipid accumulation was measured by Oil red O staining. All photographs were taken using a microscope. Lipid accumulation was inhibited by DRE (magnification ×400).

    Techniques Used: Staining, Microscopy

    Effects of fraction from Distylium racemosum on lipid accumulation in 3T3-L1 cells. (A) Hexane fraction, (B) Ethyl acetate fraction, (C) Butanol fraction, (D) Water fraction. 3T3-L1 cells were treated with the indicated concentrations (0, 12.5, 25, and 50 μg/mL) of D. racemosum fraction. Lipid accumulation was measured by Oil red O stain. The cells were eluted into 100% isopropanol and OD was measured at 500 nm. Data are expressed as mean ± standard deviation (n = 3). Significant differences from control are indicated (p
    Figure Legend Snippet: Effects of fraction from Distylium racemosum on lipid accumulation in 3T3-L1 cells. (A) Hexane fraction, (B) Ethyl acetate fraction, (C) Butanol fraction, (D) Water fraction. 3T3-L1 cells were treated with the indicated concentrations (0, 12.5, 25, and 50 μg/mL) of D. racemosum fraction. Lipid accumulation was measured by Oil red O stain. The cells were eluted into 100% isopropanol and OD was measured at 500 nm. Data are expressed as mean ± standard deviation (n = 3). Significant differences from control are indicated (p

    Techniques Used: Staining, Standard Deviation

    Effects of fraction from Distylium racemosum on 3T3-L1 cell viability. (A) Hexane fraction, (B) Ethyl acetate fraction, (C) Butanol fraction, (D) Water fraction. 3T3-L1 cells were treated with the indicated concentrations (0, 12.5, 25, 50, and 100 μg/mL) of D. racemosum fraction for 48 h. Cell viability was measured by MTT assay. Data are expressed as mean ± standard deviation (n = 3). Significant differences from control are indicated (p
    Figure Legend Snippet: Effects of fraction from Distylium racemosum on 3T3-L1 cell viability. (A) Hexane fraction, (B) Ethyl acetate fraction, (C) Butanol fraction, (D) Water fraction. 3T3-L1 cells were treated with the indicated concentrations (0, 12.5, 25, 50, and 100 μg/mL) of D. racemosum fraction for 48 h. Cell viability was measured by MTT assay. Data are expressed as mean ± standard deviation (n = 3). Significant differences from control are indicated (p

    Techniques Used: MTT Assay, Standard Deviation

    28) Product Images from "Leptin Production by Encapsulated Adipocytes Increases Brown Fat, Decreases Resistin, and Improves Glucose Intolerance in Obese Mice"

    Article Title: Leptin Production by Encapsulated Adipocytes Increases Brown Fat, Decreases Resistin, and Improves Glucose Intolerance in Obese Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153198

    Treatment with leptin-producing encapsulated adipocytes increases plasma leptin and transiently influences food intake and weight. O b/ob mice (n = 7 per group) were injected with encapsulated acellular capsules (OB [Emp] ), encapsulated 3T3-L1 preadipocytes (OB [3T3] ), and encapsulated 3T3 Lep (OB [ Lep ] ) in both visceral fat pads (total 0.6*10 6 adipocytes for each cell type). (A) Leptin concentrations in plasma from OB [Emp] (n = 4), OB [3T3] (n = 4), and OB [ Lep ] (n = 7) mice. The leptin ELISA was performed on non-hemolyzed plasma samples. All data are expressed as means ± SD. Significant differences are shown with a black bar and determined by one-way ANOVA followed by Tukey’s post hoc test ( P
    Figure Legend Snippet: Treatment with leptin-producing encapsulated adipocytes increases plasma leptin and transiently influences food intake and weight. O b/ob mice (n = 7 per group) were injected with encapsulated acellular capsules (OB [Emp] ), encapsulated 3T3-L1 preadipocytes (OB [3T3] ), and encapsulated 3T3 Lep (OB [ Lep ] ) in both visceral fat pads (total 0.6*10 6 adipocytes for each cell type). (A) Leptin concentrations in plasma from OB [Emp] (n = 4), OB [3T3] (n = 4), and OB [ Lep ] (n = 7) mice. The leptin ELISA was performed on non-hemolyzed plasma samples. All data are expressed as means ± SD. Significant differences are shown with a black bar and determined by one-way ANOVA followed by Tukey’s post hoc test ( P

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    Engineered 3T3-L1 adipocytes express and constitutively secrete leptin. 3T3-L1 cells were stably transduced to overexpress Lep (3T3 Lep ) (A, B) Lep expression in non-differentiated preadipocytes (ND = non-differentiated) (A) and differentiated preadipocytes (D = differentiated) (B) is shown for 3T3-L1 (white bar) and 3T3 Lep (black bar) (both n = 4, P
    Figure Legend Snippet: Engineered 3T3-L1 adipocytes express and constitutively secrete leptin. 3T3-L1 cells were stably transduced to overexpress Lep (3T3 Lep ) (A, B) Lep expression in non-differentiated preadipocytes (ND = non-differentiated) (A) and differentiated preadipocytes (D = differentiated) (B) is shown for 3T3-L1 (white bar) and 3T3 Lep (black bar) (both n = 4, P

    Techniques Used: Stable Transfection, Expressing

    29) Product Images from "Targeted Proteomic Analysis of Small GTPases in Murine Adipogenesis"

    Article Title: Targeted Proteomic Analysis of Small GTPases in Murine Adipogenesis

    Journal: Analytical chemistry

    doi: 10.1021/acs.analchem.0c00974

    Numbers of small GTPases quantified in 3T3-L1 and C3H10T1/2 murine cells during adipocyte differentiation. (A) 3T3-L1 murine fibroblast and C3H10T1/2 murine mesenchymal stem cells (−) were induced to differentiation into adipocytes (+). Adipogenesis efficiency was confirmed by Oil Red O staining. The lipid dye was later extracted and its UV absorbance at 500 nm was measured. The data represent the mean ± standard deviation (S.D.) of quantification results ( n = 3). (B) Venn diagram showing the numbers of small GTPases in 3T3-L1 and C3H10T1/2 cells quantified with scheduled MRM analysis. (C) Venn diagrams showing the overlap of quantified small GTPases from the two biological replicates in 3T3-L1 cells and three biological replicates in C3H10T1/2 cells.
    Figure Legend Snippet: Numbers of small GTPases quantified in 3T3-L1 and C3H10T1/2 murine cells during adipocyte differentiation. (A) 3T3-L1 murine fibroblast and C3H10T1/2 murine mesenchymal stem cells (−) were induced to differentiation into adipocytes (+). Adipogenesis efficiency was confirmed by Oil Red O staining. The lipid dye was later extracted and its UV absorbance at 500 nm was measured. The data represent the mean ± standard deviation (S.D.) of quantification results ( n = 3). (B) Venn diagram showing the numbers of small GTPases in 3T3-L1 and C3H10T1/2 cells quantified with scheduled MRM analysis. (C) Venn diagrams showing the overlap of quantified small GTPases from the two biological replicates in 3T3-L1 cells and three biological replicates in C3H10T1/2 cells.

    Techniques Used: Staining, Standard Deviation

    Role of Rab32 in adipocyte differentiation. (A) 3T3-L1 and (B) C3H10T1/2 cells were transfected with control (EGFP) or EGFP-Rab32 plasmid followed by adipogenesis induction. The undifferentiated (−) and differentiated (+) cells were stained with Oil Red O and the adipogenesis efficiency was quantified by UV adsorption at 500 nm. The p values were calculated using two-tailed, unpaired Student’s t -test: *, 0.01
    Figure Legend Snippet: Role of Rab32 in adipocyte differentiation. (A) 3T3-L1 and (B) C3H10T1/2 cells were transfected with control (EGFP) or EGFP-Rab32 plasmid followed by adipogenesis induction. The undifferentiated (−) and differentiated (+) cells were stained with Oil Red O and the adipogenesis efficiency was quantified by UV adsorption at 500 nm. The p values were calculated using two-tailed, unpaired Student’s t -test: *, 0.01

    Techniques Used: Transfection, Plasmid Preparation, Staining, Adsorption, Two Tailed Test

    Differentially expressed small GTPases upon adipocyte differentiation. (A) Heat map depicting the quantified small GTPases in 3T3-L1 and C3H10T1/2 cells upon differentiation. (B) Quantification data of top 20 differentially expressed small GTPases in 3T3-L1 and C3H10T1/2 cells. The data represent mean ± SD of results obtained from two and three biological replicates for 3T3-L1 and C3H10T1/2 cells, respectively.
    Figure Legend Snippet: Differentially expressed small GTPases upon adipocyte differentiation. (A) Heat map depicting the quantified small GTPases in 3T3-L1 and C3H10T1/2 cells upon differentiation. (B) Quantification data of top 20 differentially expressed small GTPases in 3T3-L1 and C3H10T1/2 cells. The data represent mean ± SD of results obtained from two and three biological replicates for 3T3-L1 and C3H10T1/2 cells, respectively.

    Techniques Used:

    Western blot validation of the quantification results obtained from scheduled MRM analysis. Shown are selected-ion chromatograms (upper panel) for monitoring tryptic peptides of representative small GTPases before (−) and after (+) differentiation of 3T3-L1 (A) and (B) C3H10T1/2 cells into adipocytes. The traces for the unlabeled peptides derived from cell lysates are shown in red and the corresponding traces for the spiked-in heavy isotope-labeled peptide are shown in blue. Western blot analysis (lower panel) was conducted to validate the MRM results of selected small GTPases. Band intensity of small GTPases was normalized against that of β -actin and further normalized against the ratio of the undifferentiated cells.
    Figure Legend Snippet: Western blot validation of the quantification results obtained from scheduled MRM analysis. Shown are selected-ion chromatograms (upper panel) for monitoring tryptic peptides of representative small GTPases before (−) and after (+) differentiation of 3T3-L1 (A) and (B) C3H10T1/2 cells into adipocytes. The traces for the unlabeled peptides derived from cell lysates are shown in red and the corresponding traces for the spiked-in heavy isotope-labeled peptide are shown in blue. Western blot analysis (lower panel) was conducted to validate the MRM results of selected small GTPases. Band intensity of small GTPases was normalized against that of β -actin and further normalized against the ratio of the undifferentiated cells.

    Techniques Used: Western Blot, Derivative Assay, Labeling

    30) Product Images from "Antiobesity and antidiabetic effects of the dairy bacterium Propionibacterium freudenreichii MJ2 in high-fat diet-induced obese mice by modulating lipid metabolism"

    Article Title: Antiobesity and antidiabetic effects of the dairy bacterium Propionibacterium freudenreichii MJ2 in high-fat diet-induced obese mice by modulating lipid metabolism

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-82282-5

    Effect of hkMJ2 on the expression levels of genes related to adipogenesis and lipogenesis. mRNA expression levels in 3T3-L1 adipocytes were analyzed by qPCR. 3T3-L1 preadipocytes were differentiated with differentiation-inducing medium containing MDI (IBMX + dexamethasone + insulin), and each concentration of hkMJ2 was tested. The treatment was maintained during the period of differentiation. ( A ) Preadipocyte factor-1 ( Pref-1 ) in 3T3-L1 adipocytes. ( B ) Adipogenic transcription factors ( PPARγ and C/EBPα ) in 3T3-L1 adipocytes. ( C ) Lipogenic transcription factors (FAS , SCD-1 and ACC ) in 3T3-L1 adipocytes. The data indicate the mean ± SD of three independent experiments. The p values were determined by ANOVA and Tukey’s HSD test.
    Figure Legend Snippet: Effect of hkMJ2 on the expression levels of genes related to adipogenesis and lipogenesis. mRNA expression levels in 3T3-L1 adipocytes were analyzed by qPCR. 3T3-L1 preadipocytes were differentiated with differentiation-inducing medium containing MDI (IBMX + dexamethasone + insulin), and each concentration of hkMJ2 was tested. The treatment was maintained during the period of differentiation. ( A ) Preadipocyte factor-1 ( Pref-1 ) in 3T3-L1 adipocytes. ( B ) Adipogenic transcription factors ( PPARγ and C/EBPα ) in 3T3-L1 adipocytes. ( C ) Lipogenic transcription factors (FAS , SCD-1 and ACC ) in 3T3-L1 adipocytes. The data indicate the mean ± SD of three independent experiments. The p values were determined by ANOVA and Tukey’s HSD test.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay

    Inhibitory effects of hkMJ2 and hkLP on lipid accumulation in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated by treatment with differentiation-inducing medium containing MDI (IBMX + dexamethasone + insulin), and heat-killed P. freudenreichii MJ2 (hkMJ2) or heat-killed L. plantarum (hkLP) were simultaneously administered at the designated concentrations during the period of differentiation. ( A ) Lipid droplets of the differentiated cells were visualized by oil red O staining (200× magnification) and the scale bars indicate 0.25 mm. ( B ) Lipid accumulation was quantified by measuring oil droplets stained with oil red O and comparing them with cells treated with MDI alone. The data indicate the mean ± SD of three independent experiments. The p values are determined by ANOVA and Tukey’s HSD test.
    Figure Legend Snippet: Inhibitory effects of hkMJ2 and hkLP on lipid accumulation in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated by treatment with differentiation-inducing medium containing MDI (IBMX + dexamethasone + insulin), and heat-killed P. freudenreichii MJ2 (hkMJ2) or heat-killed L. plantarum (hkLP) were simultaneously administered at the designated concentrations during the period of differentiation. ( A ) Lipid droplets of the differentiated cells were visualized by oil red O staining (200× magnification) and the scale bars indicate 0.25 mm. ( B ) Lipid accumulation was quantified by measuring oil droplets stained with oil red O and comparing them with cells treated with MDI alone. The data indicate the mean ± SD of three independent experiments. The p values are determined by ANOVA and Tukey’s HSD test.

    Techniques Used: Staining

    31) Product Images from "DMRT2 Interacts With FXR and Improves Insulin Resistance in Adipocytes and a Mouse Model"

    Article Title: DMRT2 Interacts With FXR and Improves Insulin Resistance in Adipocytes and a Mouse Model

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2021.723623

    DMRT2 is downregulated in insulin-resistant adipocyte model. (A) 3T3-L1 preadipocytes were induced toward adipogenesis and the formation of lip droplets in preadipocytes and adipocytes was confirmed using oil O red staining. (B) The mRNA expression levels of Fabp4 and PPARγ in preadipocytes and adipocytes were examined using qRT-PCR. Then, the insulin-resistant adipocyte model was established in differentiated adipocytes as described and examined for Fabp4 and PPARγ mRNA levels (C) , glucose uptake ability (D) , and triglyceride content (E) ; cellular membrane-located GLUT4 levels in control or insulin-resistant (IR) adipocytes by immunofluorescent (IF) staining (F) ; membrane protein levels of GLUT4 in control or IR adipocytes by immunoblotting (G) ; the protein levels of Akt and p-Akt in control or IR adipocytes by immunoblotting (H) ; the mRNA expression of TNF-α and IL-6 in control or IR adipocytes by qRT-PCR (I) ; the secretion levels of TNF-α and IL-6 in control or IR adipocytes by ELISA (J) ; and the protein levels of DMRT2 in control or IR adipocytes by immunoblotting (K) . * P
    Figure Legend Snippet: DMRT2 is downregulated in insulin-resistant adipocyte model. (A) 3T3-L1 preadipocytes were induced toward adipogenesis and the formation of lip droplets in preadipocytes and adipocytes was confirmed using oil O red staining. (B) The mRNA expression levels of Fabp4 and PPARγ in preadipocytes and adipocytes were examined using qRT-PCR. Then, the insulin-resistant adipocyte model was established in differentiated adipocytes as described and examined for Fabp4 and PPARγ mRNA levels (C) , glucose uptake ability (D) , and triglyceride content (E) ; cellular membrane-located GLUT4 levels in control or insulin-resistant (IR) adipocytes by immunofluorescent (IF) staining (F) ; membrane protein levels of GLUT4 in control or IR adipocytes by immunoblotting (G) ; the protein levels of Akt and p-Akt in control or IR adipocytes by immunoblotting (H) ; the mRNA expression of TNF-α and IL-6 in control or IR adipocytes by qRT-PCR (I) ; the secretion levels of TNF-α and IL-6 in control or IR adipocytes by ELISA (J) ; and the protein levels of DMRT2 in control or IR adipocytes by immunoblotting (K) . * P

    Techniques Used: Staining, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    DMRT2 directly interacts with FXR. (A) Protein–protein interaction analysis showing proteins that might interact with DMRT2. (B) The protein levels of FXR in IR adipocytes and IF adipocytes were examined using immunoblotting. (C) IR adipocytes were transfected with DMRT2 OE or sh-DMRT2 and examined for the protein levels of FXR by immunoblotting. (D) The interaction between DMRT2 and FXR was examined using IF staining. Green fluorescence indicated DMRT2. Red fluorescence indicated FXR. (E, F) The interaction between DMRT2 and FXR was examined using the co-IP assay. (G) 3T3-L1 cells were co-transfected with DMRT2 overexpression vector and GLUT4 promoter luciferase reporter vector. Then, the luciferase activity was measured. * P
    Figure Legend Snippet: DMRT2 directly interacts with FXR. (A) Protein–protein interaction analysis showing proteins that might interact with DMRT2. (B) The protein levels of FXR in IR adipocytes and IF adipocytes were examined using immunoblotting. (C) IR adipocytes were transfected with DMRT2 OE or sh-DMRT2 and examined for the protein levels of FXR by immunoblotting. (D) The interaction between DMRT2 and FXR was examined using IF staining. Green fluorescence indicated DMRT2. Red fluorescence indicated FXR. (E, F) The interaction between DMRT2 and FXR was examined using the co-IP assay. (G) 3T3-L1 cells were co-transfected with DMRT2 overexpression vector and GLUT4 promoter luciferase reporter vector. Then, the luciferase activity was measured. * P

    Techniques Used: Transfection, Staining, Fluorescence, Co-Immunoprecipitation Assay, Over Expression, Plasmid Preparation, Luciferase, Activity Assay

    32) Product Images from "Triphenyl phosphate is a selective PPARγ modulator that does not induce brite adipogenesis in vitro and in vivo"

    Article Title: Triphenyl phosphate is a selective PPARγ modulator that does not induce brite adipogenesis in vitro and in vivo

    Journal: bioRxiv

    doi: 10.1101/626390

    Proteomes of 3T3-L1 adipocytes differentiated with Rosi and TPhP. Confluent 3T3-L1 cells were differentiated as described in Fig. 3 . The proteome was analyzed by precision quantitative nanoLC-tandem MS. a Heatmap and (b) volcano plot of top differentially expressed proteins between Rosi and TPhP.
    Figure Legend Snippet: Proteomes of 3T3-L1 adipocytes differentiated with Rosi and TPhP. Confluent 3T3-L1 cells were differentiated as described in Fig. 3 . The proteome was analyzed by precision quantitative nanoLC-tandem MS. a Heatmap and (b) volcano plot of top differentially expressed proteins between Rosi and TPhP.

    Techniques Used:

    Phosphoproteomes of 3T3-L1 adipocytes differentiated with Rosi and TPhP. Confluent 3T3-L1 cells were differentiated as described in Fig. 3 . Phospho-peptides were enriched using TiO2 and then analyzed by precision quantitative nanoLC-tandem MS. a Heatmap and (b) volcano plot of top differentially expressed phospho-proteins between Rosi and TPhP.
    Figure Legend Snippet: Phosphoproteomes of 3T3-L1 adipocytes differentiated with Rosi and TPhP. Confluent 3T3-L1 cells were differentiated as described in Fig. 3 . Phospho-peptides were enriched using TiO2 and then analyzed by precision quantitative nanoLC-tandem MS. a Heatmap and (b) volcano plot of top differentially expressed phospho-proteins between Rosi and TPhP.

    Techniques Used:

    PPARγ phosphorylation and its effect on gene expression in mouse adipocytes differentiated with Rosi and TPhP. a Confluent 3T3-L1 cells were differentiated as described in Fig. 3 and phosphorylation of PPARγ at ser273 was determined relative to total PPARγ by immunoblot. b Confluent 3T3-L1 cells were differentiated as described in Fig. 3 , in the presence or absence of roscovitine (Rosco, 20 μM). Gene expression was determined by RT-qPCR and presented as a heatmap of expression levels of white adipocyte and brite adipocyte marker genes. c Confluent 3T3 cells expressing wildtype PPARγ (3T3-WT) or PPARγ with alanine substituted for serine 273 (3T3-SA) were differentiated as described for 3T3-L1 cells in Fig. 3 . Gene expression was determined by RT-qPCR and presented as a heatmap of expression levels of white and brite adipocyte marker genes. Data are presented as mean ± SE (n≥3). Statistically different from Vh-treated (*p
    Figure Legend Snippet: PPARγ phosphorylation and its effect on gene expression in mouse adipocytes differentiated with Rosi and TPhP. a Confluent 3T3-L1 cells were differentiated as described in Fig. 3 and phosphorylation of PPARγ at ser273 was determined relative to total PPARγ by immunoblot. b Confluent 3T3-L1 cells were differentiated as described in Fig. 3 , in the presence or absence of roscovitine (Rosco, 20 μM). Gene expression was determined by RT-qPCR and presented as a heatmap of expression levels of white adipocyte and brite adipocyte marker genes. c Confluent 3T3 cells expressing wildtype PPARγ (3T3-WT) or PPARγ with alanine substituted for serine 273 (3T3-SA) were differentiated as described for 3T3-L1 cells in Fig. 3 . Gene expression was determined by RT-qPCR and presented as a heatmap of expression levels of white and brite adipocyte marker genes. Data are presented as mean ± SE (n≥3). Statistically different from Vh-treated (*p

    Techniques Used: Expressing, Quantitative RT-PCR, Marker

    Functions of and gene expression in 3T3-L1 adipocytes differentiated with Rosi and TPhP. Confluent 3T3-L1 cells were differentiated using a standard hormone cocktail for 10 days. During differentiation, cells were treated with Vh (0.1% DMSO, final concentration), Rosi (20 μM), or TPhP (20 μM) in the presence or absence of the PPARγ antagonist T0070907 (20 µM). a Lipid accumulation was determined by Nile Red staining. b Gene expression was analyzed by RT-qPCR. c Mitochondrial biogenesis was analyzed by measuring mitochondria-specific proteins. d Cellular respiration was measured using the Seahorse assay. Data are presented as mean ± SE (n=3-6). Statistically different from Vh-treated (*p
    Figure Legend Snippet: Functions of and gene expression in 3T3-L1 adipocytes differentiated with Rosi and TPhP. Confluent 3T3-L1 cells were differentiated using a standard hormone cocktail for 10 days. During differentiation, cells were treated with Vh (0.1% DMSO, final concentration), Rosi (20 μM), or TPhP (20 μM) in the presence or absence of the PPARγ antagonist T0070907 (20 µM). a Lipid accumulation was determined by Nile Red staining. b Gene expression was analyzed by RT-qPCR. c Mitochondrial biogenesis was analyzed by measuring mitochondria-specific proteins. d Cellular respiration was measured using the Seahorse assay. Data are presented as mean ± SE (n=3-6). Statistically different from Vh-treated (*p

    Techniques Used: Expressing, Concentration Assay, Staining, Quantitative RT-PCR

    33) Product Images from "ACSS3 in brown fat drives propionate catabolism and its deficiency leads to autophagy and systemic metabolic dysfunction, et al. ACSS3 in brown fat drives propionate catabolism and its deficiency leads to autophagy and systemic metabolic dysfunction"

    Article Title: ACSS3 in brown fat drives propionate catabolism and its deficiency leads to autophagy and systemic metabolic dysfunction, et al. ACSS3 in brown fat drives propionate catabolism and its deficiency leads to autophagy and systemic metabolic dysfunction

    Journal: Clinical and Translational Medicine

    doi: 10.1002/ctm2.665

    Propionate promotes autophagy of adipocytes. (A) Western blot analysis of LC3 (upper) and ratios of LC3 II/I (lower) of mouse BAT cells treated with 3 mM sodium acetate (acetate), sodium propionate (propionate) and sodium butyrate (butyrate). N = 3 independent treatments. (B) Staining of P62 (green) of saline‐ and propionate‐treated BAT cells. Scale bar: 10 μm. (C) Co‐staining of LC3 (green) and LAMP2 (red) in 8‐day differentiated preadipocytes isolated from BAT of WT mice treated with saline and propionate. White arrowheads indicate the co‐localisation. Scale bar: 20 μm. (D) Relative levels of Lc3b , Atg5 , Atg7 , Ulk1 and Beclin1 from saline‐ and propionate‐treated 6‐day differentiated BAT cell line; N = 4 independent treatments. (E, F) Relative levels of Pparγ, Fabp4 , Srebp1 , Atg5 , Ulk1 and Beclin1 from saline‐ and propionate‐treated 8‐day differentiated SVF preadipocytes isolated from iWAT (E) and eWAT (F) of 8‐week‐old WT mice; N = 4 independent treatments. (G) Western blot analysis of LC3 of differentiated 3T3‐L1 cells treated with 3 mM sodium acetate (acetate), sodium propionate (propionate) and sodium butyrate (butyrate). Rapamycin‐treated cells were set as positive control. Numbers below the blot indicate ratios of LC3 II/I. (H) Western blot analysis of P62 from differentiated human A41 preadipocytes treated with 3 mM acetate, propionate and butyrate; numbers below the blot indicate ratio of P62/GAPDH. (I) P62 (green) staining of saline and propionate treated differentiated human A41 preadipocytes. Scale bar: 20 μm. (J) Co‐staining of LC3 (green) and LAMP2 (red) in 14‐day differentiated human A41 preadipocytes treated with saline and propionate. White arrowheads indicate the co‐localisation. Scale bar: 20 μm. (K) Bodipy staining indicates enlarged LDs of propionate‐treated human white adipocytes. White arrowheads indicate the larger LDs after propionate treatment. Scale bar: 20 μm. (L) Western blot analysis of p‐4E‐BP1 (Thr37/46) of human A41 adipocytes treated with 0.3 mM sodium propionate. N = 3 independent treatments. Data represent mean ± SEM ( t ‐test: * p
    Figure Legend Snippet: Propionate promotes autophagy of adipocytes. (A) Western blot analysis of LC3 (upper) and ratios of LC3 II/I (lower) of mouse BAT cells treated with 3 mM sodium acetate (acetate), sodium propionate (propionate) and sodium butyrate (butyrate). N = 3 independent treatments. (B) Staining of P62 (green) of saline‐ and propionate‐treated BAT cells. Scale bar: 10 μm. (C) Co‐staining of LC3 (green) and LAMP2 (red) in 8‐day differentiated preadipocytes isolated from BAT of WT mice treated with saline and propionate. White arrowheads indicate the co‐localisation. Scale bar: 20 μm. (D) Relative levels of Lc3b , Atg5 , Atg7 , Ulk1 and Beclin1 from saline‐ and propionate‐treated 6‐day differentiated BAT cell line; N = 4 independent treatments. (E, F) Relative levels of Pparγ, Fabp4 , Srebp1 , Atg5 , Ulk1 and Beclin1 from saline‐ and propionate‐treated 8‐day differentiated SVF preadipocytes isolated from iWAT (E) and eWAT (F) of 8‐week‐old WT mice; N = 4 independent treatments. (G) Western blot analysis of LC3 of differentiated 3T3‐L1 cells treated with 3 mM sodium acetate (acetate), sodium propionate (propionate) and sodium butyrate (butyrate). Rapamycin‐treated cells were set as positive control. Numbers below the blot indicate ratios of LC3 II/I. (H) Western blot analysis of P62 from differentiated human A41 preadipocytes treated with 3 mM acetate, propionate and butyrate; numbers below the blot indicate ratio of P62/GAPDH. (I) P62 (green) staining of saline and propionate treated differentiated human A41 preadipocytes. Scale bar: 20 μm. (J) Co‐staining of LC3 (green) and LAMP2 (red) in 14‐day differentiated human A41 preadipocytes treated with saline and propionate. White arrowheads indicate the co‐localisation. Scale bar: 20 μm. (K) Bodipy staining indicates enlarged LDs of propionate‐treated human white adipocytes. White arrowheads indicate the larger LDs after propionate treatment. Scale bar: 20 μm. (L) Western blot analysis of p‐4E‐BP1 (Thr37/46) of human A41 adipocytes treated with 0.3 mM sodium propionate. N = 3 independent treatments. Data represent mean ± SEM ( t ‐test: * p

    Techniques Used: Western Blot, Staining, Isolation, Mouse Assay, Positive Control

    34) Product Images from "The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPARγ"

    Article Title: The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPARγ

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0162-6

    Negative correlation between TRIM25 and PPARγ expression both in mice and humans. a Immunoblot analysis of TRIM25, PPARγ, and adipogenic markers during 3T3-L1 cell differentiation. Relative TRIM25 protein levels were measured using ImageJ (bottom graphs). b Real-time quantitative PCR analysis of the mRNA expression of TRIM25, PPARγ, and adipogenic markers during 3T3-L1 cell differentiation. c Immunoblot analysis of TRIM25 and PPARγ in white adipose tissue (WAT) of normal chow-fed (NC) vs. high-fat-fed (HFD) mice. d Immunoblot analysis of TRIM25 and PPARγ in WAT of control vs. ob/ob mice. Relative TRIM25 and PPARγ protein levels were measured using ImageJ (right graphs). e Pearson correlation coefficients between TRIM25 and PPARγ levels within human subcutaneous fat samples were calculated. All error bars shown are the s.e.m. (* p
    Figure Legend Snippet: Negative correlation between TRIM25 and PPARγ expression both in mice and humans. a Immunoblot analysis of TRIM25, PPARγ, and adipogenic markers during 3T3-L1 cell differentiation. Relative TRIM25 protein levels were measured using ImageJ (bottom graphs). b Real-time quantitative PCR analysis of the mRNA expression of TRIM25, PPARγ, and adipogenic markers during 3T3-L1 cell differentiation. c Immunoblot analysis of TRIM25 and PPARγ in white adipose tissue (WAT) of normal chow-fed (NC) vs. high-fat-fed (HFD) mice. d Immunoblot analysis of TRIM25 and PPARγ in WAT of control vs. ob/ob mice. Relative TRIM25 and PPARγ protein levels were measured using ImageJ (right graphs). e Pearson correlation coefficients between TRIM25 and PPARγ levels within human subcutaneous fat samples were calculated. All error bars shown are the s.e.m. (* p

    Techniques Used: Expressing, Mouse Assay, Cell Differentiation, Real-time Polymerase Chain Reaction

    TRIM25 suppresses adipocyte differentiation in 3T3-L1 cells. Stably expression of TRIM25 WT and TRIM25 CS ( a – c ) or shRNAs (#1 and #2) for TRIM25 ( d – f ) in 3T3-L1 pre-adipocytes was induced for 6 days. a , d Differentiated cells were stained by Oil Red O. b , e The protein expression of TRIM25, PPARγ, and adipogenic markers was analyzed by western blotting. c , f The mRNA expression of adipogenic markers was analyzed by real-time quantitative PCR. All error bars shown are the s.e.m. (* p
    Figure Legend Snippet: TRIM25 suppresses adipocyte differentiation in 3T3-L1 cells. Stably expression of TRIM25 WT and TRIM25 CS ( a – c ) or shRNAs (#1 and #2) for TRIM25 ( d – f ) in 3T3-L1 pre-adipocytes was induced for 6 days. a , d Differentiated cells were stained by Oil Red O. b , e The protein expression of TRIM25, PPARγ, and adipogenic markers was analyzed by western blotting. c , f The mRNA expression of adipogenic markers was analyzed by real-time quantitative PCR. All error bars shown are the s.e.m. (* p

    Techniques Used: Stable Transfection, Expressing, Staining, Western Blot, Real-time Polymerase Chain Reaction

    TRIM25 mediates the ubiquitination of PPARγ through its E3 ligase activity. a 3T3-L1 cells were stably expressed with TRIM25 WT or TRIM25 CS and differentiated for 6 days. Cells were treated with MG132 for 6 h. Cell lysates were incubated with an α-PPARγ antibody, and ubiquitination of PPARγ was analyzed by western blotting. b HEK-293 cells were transfected with TRIM25 WT or TRIM25 CS . Cell lysates were incubated with an α-FLAG antibody, and ubiquitination of PPARγ was analyzed by western blotting. c Ubiquitination of PPARγ by TRIM25 in vitro. Purified GST-tagged PPARγ was incubated with E1, E2, ubiquitin (Ub), TRIM25 WT , or TRIM25 CS in the absence and presence of ATP as indicated. Ubiquitination of PPARγ was analyzed by western blotting using the indicated antibodies
    Figure Legend Snippet: TRIM25 mediates the ubiquitination of PPARγ through its E3 ligase activity. a 3T3-L1 cells were stably expressed with TRIM25 WT or TRIM25 CS and differentiated for 6 days. Cells were treated with MG132 for 6 h. Cell lysates were incubated with an α-PPARγ antibody, and ubiquitination of PPARγ was analyzed by western blotting. b HEK-293 cells were transfected with TRIM25 WT or TRIM25 CS . Cell lysates were incubated with an α-FLAG antibody, and ubiquitination of PPARγ was analyzed by western blotting. c Ubiquitination of PPARγ by TRIM25 in vitro. Purified GST-tagged PPARγ was incubated with E1, E2, ubiquitin (Ub), TRIM25 WT , or TRIM25 CS in the absence and presence of ATP as indicated. Ubiquitination of PPARγ was analyzed by western blotting using the indicated antibodies

    Techniques Used: Activity Assay, Stable Transfection, Incubation, Western Blot, Transfection, In Vitro, Purification

    35) Product Images from "Differentiated Embryo Chondrocyte 1 (DEC1) Represses PPAR?2 Gene through Interacting with CCAAT/Enhancer Binding Protein ? (C/EBP?)"

    Article Title: Differentiated Embryo Chondrocyte 1 (DEC1) Represses PPAR?2 Gene through Interacting with CCAAT/Enhancer Binding Protein ? (C/EBP?)

    Journal: Molecules and Cells

    doi: 10.1007/s10059-012-0002-9

    ). The 18S rRNA was used as a loading control. (C) Adipogenesis was induced by MDI treatment in HIF-1α knockdown 3T3-L1 ( sh HIF-1α) cells and control 3T3-L1 ( sh Control) cells for the indicated times. DEC1, HIF-1α, and C/EBPβ protein levels were detected by western analyses. Tubulin was detected to check for equal loadings.
    Figure Legend Snippet: ). The 18S rRNA was used as a loading control. (C) Adipogenesis was induced by MDI treatment in HIF-1α knockdown 3T3-L1 ( sh HIF-1α) cells and control 3T3-L1 ( sh Control) cells for the indicated times. DEC1, HIF-1α, and C/EBPβ protein levels were detected by western analyses. Tubulin was detected to check for equal loadings.

    Techniques Used: Western Blot

    Effects of DEC1 on the activity of C/EBPβ. (A) 3xC/EBP-Luc reporter plasmid and the indicated plasmids were transfected into 3T3-L1 cells with pCHO110. After 24 h, the transfected cells were further incubated under either normoxic or hypoxic conditions (0.1–0.5% oxygen) for 24 h before harvest. The luciferase activity of the cell lysate was measured and normalized based on β-galactosidase activity as described previously. (B) The PPARγ2 -Luc reporter plasmids and the indicated plasmids were transfected into 3T3-L1 cells with the pRL-TK plasmids as described above. The luciferase activity was measured and normalized based on the Renilla luciferase activity. The values indicated averages and standard deviations of three independent experiments. (C) 3T3-L1 cells were transfected with si RNA against DEC1 as described. Before harvest, the transfected cells were exposed to hypoxia (0.1–0.5% oxygen) for 24 h. The levels of DEC1 mRNA were quantified by qRT-PCR. (D) The PPARγ2 -Luc reporter plasmids were transfected with the indicated plasmid or si RNA into 3T3-L1 cells. The transfected cells were incubated in hypoxia (0.1–0.5% oxygen) for 24 h before harvesting, and luciferase assays were performed. Values represent the means and standard deviations of three experiments. P values for the indicated sets are calculated by Student’s t -test; * p
    Figure Legend Snippet: Effects of DEC1 on the activity of C/EBPβ. (A) 3xC/EBP-Luc reporter plasmid and the indicated plasmids were transfected into 3T3-L1 cells with pCHO110. After 24 h, the transfected cells were further incubated under either normoxic or hypoxic conditions (0.1–0.5% oxygen) for 24 h before harvest. The luciferase activity of the cell lysate was measured and normalized based on β-galactosidase activity as described previously. (B) The PPARγ2 -Luc reporter plasmids and the indicated plasmids were transfected into 3T3-L1 cells with the pRL-TK plasmids as described above. The luciferase activity was measured and normalized based on the Renilla luciferase activity. The values indicated averages and standard deviations of three independent experiments. (C) 3T3-L1 cells were transfected with si RNA against DEC1 as described. Before harvest, the transfected cells were exposed to hypoxia (0.1–0.5% oxygen) for 24 h. The levels of DEC1 mRNA were quantified by qRT-PCR. (D) The PPARγ2 -Luc reporter plasmids were transfected with the indicated plasmid or si RNA into 3T3-L1 cells. The transfected cells were incubated in hypoxia (0.1–0.5% oxygen) for 24 h before harvesting, and luciferase assays were performed. Values represent the means and standard deviations of three experiments. P values for the indicated sets are calculated by Student’s t -test; * p

    Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Incubation, Luciferase, Quantitative RT-PCR

    36) Product Images from "Actions of β-Apo-Carotenoids in Differentiating Cells: Differential Effects in P19 Cells and 3T3-L1 Adipocytes"

    Article Title: Actions of β-Apo-Carotenoids in Differentiating Cells: Differential Effects in P19 Cells and 3T3-L1 Adipocytes

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2015.01.009

    Short-term (1 day) treatment of differentiating 3T3-L1 adipocytes early in the differentiation program with β-apo-13-carotenone stimulates 3T3-L1 adipocyte differentiation as assessed by adipocyte-specific gene expression As described in the text, the culture medium of differentiating 3T3-L1 adipocytes was supplemented for 1 day with either vehicle alone (Ctrl), all- trans -retinoic acid (0.1 μM final concentration), β-apo-13-carotenone (5 μM), or a combination of all- trans -retinoic acid (0.1 μM) and β-apo-13-carotenone (5 μM). Subsequently, expression levels of 4 adipocyte-specific genes (lipoprotein lipase (Lpl), Ap2, adiponectin, and Pparg) were assessed by qPCR. All data are normalized to Gapdh mRNA levels. Values are presented as means ± SEM (n = 3). Expression levels that are denoted with different letters are significantly different (P
    Figure Legend Snippet: Short-term (1 day) treatment of differentiating 3T3-L1 adipocytes early in the differentiation program with β-apo-13-carotenone stimulates 3T3-L1 adipocyte differentiation as assessed by adipocyte-specific gene expression As described in the text, the culture medium of differentiating 3T3-L1 adipocytes was supplemented for 1 day with either vehicle alone (Ctrl), all- trans -retinoic acid (0.1 μM final concentration), β-apo-13-carotenone (5 μM), or a combination of all- trans -retinoic acid (0.1 μM) and β-apo-13-carotenone (5 μM). Subsequently, expression levels of 4 adipocyte-specific genes (lipoprotein lipase (Lpl), Ap2, adiponectin, and Pparg) were assessed by qPCR. All data are normalized to Gapdh mRNA levels. Values are presented as means ± SEM (n = 3). Expression levels that are denoted with different letters are significantly different (P

    Techniques Used: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction

    β-Apo-13-carotenone treatment of differentiating 3T3-L1 adipocytes for 7 days does not stimulate adipocyte-specific gene expression or arrest the inhibitory effects of all- trans -retinoic acid on differentiation As described in the text, the culture medium of differentiating 3T3-L1 adipocytes was supplemented for 7 days with either vehicle alone (Ctrl), all- trans -retinoic acid (0.1 μM final concentration), β-apo-13-carotenone (5 μM), or a combination of all- trans -retinoic acid (0.1 μM) and μ-apo-13-carotenone (5 μM). Subsequently, expression levels of 3 adipocyte-specific genes (lipoprotein lipase (Lpl), Ap2 and adiponectin) were assessed by qPCR. All data are normalized to Gapdh mRNA levels. Values are presented as means ± SEM (n = 3). Expression levels that are denoted with different letters are significantly different (P
    Figure Legend Snippet: β-Apo-13-carotenone treatment of differentiating 3T3-L1 adipocytes for 7 days does not stimulate adipocyte-specific gene expression or arrest the inhibitory effects of all- trans -retinoic acid on differentiation As described in the text, the culture medium of differentiating 3T3-L1 adipocytes was supplemented for 7 days with either vehicle alone (Ctrl), all- trans -retinoic acid (0.1 μM final concentration), β-apo-13-carotenone (5 μM), or a combination of all- trans -retinoic acid (0.1 μM) and μ-apo-13-carotenone (5 μM). Subsequently, expression levels of 3 adipocyte-specific genes (lipoprotein lipase (Lpl), Ap2 and adiponectin) were assessed by qPCR. All data are normalized to Gapdh mRNA levels. Values are presented as means ± SEM (n = 3). Expression levels that are denoted with different letters are significantly different (P

    Techniques Used: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction

    37) Product Images from "TRIM47 is a novel endothelial activation factor that aggravates lipopolysaccharide-induced acute lung injury in mice via K63-linked ubiquitination of TRAF2"

    Article Title: TRIM47 is a novel endothelial activation factor that aggravates lipopolysaccharide-induced acute lung injury in mice via K63-linked ubiquitination of TRAF2

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-022-00953-9

    TRIM47 is highly expressed in vascular endothelial cells. a The relative expression of TRIM47 gene in different cells was detected by real-time PCR ( n = 3). b Representative images of the immunoblots of TIRM47 and β-actin in different cells detected by western blot (up) and the relative quantitation of TIRM47 protein levels (down, n = 3). THP-1, human monocytic cell line, RAW264.7, murine macrophage cell line, HL-1, mouse cardiac muscle cell line, HUVEC, human umbilical vein endothelial cells, EA.hy926, human umbilical vein cell line, hCMEC/D3, human brain microvascular endothelial cell line, bEnd.3, mouse brain microvascular endothelial cell line, HEK293, Human embryonic kidney 293 cells, U251, human glioma cell line, HeLa, human cervical cancer cells, MDA-MB-231, human triple-negative breast cancer cell line, A549, human lung carcinoma cell line, and 3T3-L1, mouse preadipocytes. c Representative immunohistochemical images showing positive staining of TRIM47 in the vascular lining of multiple tissues (partial amplification from Fig. 1a , n = 3). Scale bar, 50 μm. The relative expression of 56 TRIM genes in ( d ) HUVECs and ( e ) hCMEC/D3 was detected by real-time PCR. ND: not detected ( n = 3)
    Figure Legend Snippet: TRIM47 is highly expressed in vascular endothelial cells. a The relative expression of TRIM47 gene in different cells was detected by real-time PCR ( n = 3). b Representative images of the immunoblots of TIRM47 and β-actin in different cells detected by western blot (up) and the relative quantitation of TIRM47 protein levels (down, n = 3). THP-1, human monocytic cell line, RAW264.7, murine macrophage cell line, HL-1, mouse cardiac muscle cell line, HUVEC, human umbilical vein endothelial cells, EA.hy926, human umbilical vein cell line, hCMEC/D3, human brain microvascular endothelial cell line, bEnd.3, mouse brain microvascular endothelial cell line, HEK293, Human embryonic kidney 293 cells, U251, human glioma cell line, HeLa, human cervical cancer cells, MDA-MB-231, human triple-negative breast cancer cell line, A549, human lung carcinoma cell line, and 3T3-L1, mouse preadipocytes. c Representative immunohistochemical images showing positive staining of TRIM47 in the vascular lining of multiple tissues (partial amplification from Fig. 1a , n = 3). Scale bar, 50 μm. The relative expression of 56 TRIM genes in ( d ) HUVECs and ( e ) hCMEC/D3 was detected by real-time PCR. ND: not detected ( n = 3)

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay, Multiple Displacement Amplification, Immunohistochemistry, Staining, Amplification

    38) Product Images from "Latent Transforming Growth Factor β Binding Protein 3 Controls Adipogenesis"

    Article Title: Latent Transforming Growth Factor β Binding Protein 3 Controls Adipogenesis

    Journal: bioRxiv

    doi: 10.1101/2022.07.10.499446

    Effect of Ltbp3 downregulation in 3T3-L1, 10T1/2, M2, and BMSC. ( A - H) Relative transcript levels of Ltbp3 and Pparγ measured at 96 h post siCtrl or siLtbp3-2 treatment (48 h in basal media followed by 48 h of adipogenic stimulation except for BMSC, BMSC were exposed to adipogenic media for 72 h) with 3T3-L1 ( A and B ), M2 ( C and D ), 10T1/2 ( E and F ), and BMSC ( G and H ) cells. For Fig. A-F, data are the mean ± SEM of 3 independent experiments with one-three technical replicas for each experiment and treatment condition. Each technical replica was analyzed two times by qRT-PCR. For experiments G and H , there were 5-6 animals per group and the samples were each assayed in duplicate by qRT-PCR. Statistical significance was evaluated by nonparametric, Mann-Whitney U test ( A - H) . *p
    Figure Legend Snippet: Effect of Ltbp3 downregulation in 3T3-L1, 10T1/2, M2, and BMSC. ( A - H) Relative transcript levels of Ltbp3 and Pparγ measured at 96 h post siCtrl or siLtbp3-2 treatment (48 h in basal media followed by 48 h of adipogenic stimulation except for BMSC, BMSC were exposed to adipogenic media for 72 h) with 3T3-L1 ( A and B ), M2 ( C and D ), 10T1/2 ( E and F ), and BMSC ( G and H ) cells. For Fig. A-F, data are the mean ± SEM of 3 independent experiments with one-three technical replicas for each experiment and treatment condition. Each technical replica was analyzed two times by qRT-PCR. For experiments G and H , there were 5-6 animals per group and the samples were each assayed in duplicate by qRT-PCR. Statistical significance was evaluated by nonparametric, Mann-Whitney U test ( A - H) . *p

    Techniques Used: Quantitative RT-PCR, MANN-WHITNEY

    39) Product Images from "FATP1 Exerts Variable Effects on Adipogenic Differentiation and Proliferation in Cells Derived From Muscle and Adipose Tissue"

    Article Title: FATP1 Exerts Variable Effects on Adipogenic Differentiation and Proliferation in Cells Derived From Muscle and Adipose Tissue

    Journal: Frontiers in Veterinary Science

    doi: 10.3389/fvets.2022.904879

    FATP1 inhibits both the proliferation and adipogenic differentiation of 3T3-L1 cells. (A) The overexpression efficiency of PPARG and FATP1 and (G) the interference efficiency in 3T3-L1 cells determined by RT-qPCR. Histogram showing the quantitation of oil red O staining after FATP1 overexpression (B) and FATP1 interference (H) . The triglyceride levels on day 8 of adipogenic differentiation in 3T3-L1 cells after FATP1 overexpression (C) and FATPT interference (I) . Micrographs of EdU assays in 3T3-L1 cells after FATP1 overexpression (D) and FATP1 interference (J) . Scale bar, 200 μm. Statistical analysis of EdU assays in 3T3-L1 cells after FATP1 overexpression (E) and FATP1 interference (K) . Statistical analysis of CCK-8 assays in 3T3-L1 cells after FATP1 overexpression (F) and FATP1 interference (L) . Data are presented as means ± SD, * p
    Figure Legend Snippet: FATP1 inhibits both the proliferation and adipogenic differentiation of 3T3-L1 cells. (A) The overexpression efficiency of PPARG and FATP1 and (G) the interference efficiency in 3T3-L1 cells determined by RT-qPCR. Histogram showing the quantitation of oil red O staining after FATP1 overexpression (B) and FATP1 interference (H) . The triglyceride levels on day 8 of adipogenic differentiation in 3T3-L1 cells after FATP1 overexpression (C) and FATPT interference (I) . Micrographs of EdU assays in 3T3-L1 cells after FATP1 overexpression (D) and FATP1 interference (J) . Scale bar, 200 μm. Statistical analysis of EdU assays in 3T3-L1 cells after FATP1 overexpression (E) and FATP1 interference (K) . Statistical analysis of CCK-8 assays in 3T3-L1 cells after FATP1 overexpression (F) and FATP1 interference (L) . Data are presented as means ± SD, * p

    Techniques Used: Over Expression, Quantitative RT-PCR, Quantitation Assay, Staining, CCK-8 Assay

    The mRNA expression profile of FATP1 in tissues and cells as determined by RT-qPCR. (A) The tissue expression profile of FATP1 in the buffalo. (B–D) Expression dynamics of FATP1 during adipogenic differentiation in buffalo myocytes, intramuscular preadipocytes, and subcutaneous preadipocytes. (E,F) Expression dynamics of FATP1 during adipogenic differentiation in mouse 3T3-L1 and C2C12 cells. Data are presented as means ± SD; lowercase letters indicate a significant difference ( p
    Figure Legend Snippet: The mRNA expression profile of FATP1 in tissues and cells as determined by RT-qPCR. (A) The tissue expression profile of FATP1 in the buffalo. (B–D) Expression dynamics of FATP1 during adipogenic differentiation in buffalo myocytes, intramuscular preadipocytes, and subcutaneous preadipocytes. (E,F) Expression dynamics of FATP1 during adipogenic differentiation in mouse 3T3-L1 and C2C12 cells. Data are presented as means ± SD; lowercase letters indicate a significant difference ( p

    Techniques Used: Expressing, Quantitative RT-PCR

    40) Product Images from "Cornus mas L. Increases Glucose Uptake and the Expression of PPARG in Insulin-Resistant Adipocytes"

    Article Title: Cornus mas L. Increases Glucose Uptake and the Expression of PPARG in Insulin-Resistant Adipocytes

    Journal: Nutrients

    doi: 10.3390/nu14112307

    Glucose uptake test in control adipocytes and adipocytes with insulin resistance (induced using palmitic acid, 16:0) at baseline and after insulin stimulation (INS), and in IR adipocytes treated with red and yellow CM fruit extract at the concentration of 0.01 mg/mL in: ( A ) 3T3-L1 adipocytes, ( B ) SAT-derived human adipocytes, ( C ) VAT-derived human adipocytes. * p
    Figure Legend Snippet: Glucose uptake test in control adipocytes and adipocytes with insulin resistance (induced using palmitic acid, 16:0) at baseline and after insulin stimulation (INS), and in IR adipocytes treated with red and yellow CM fruit extract at the concentration of 0.01 mg/mL in: ( A ) 3T3-L1 adipocytes, ( B ) SAT-derived human adipocytes, ( C ) VAT-derived human adipocytes. * p

    Techniques Used: Concentration Assay, Derivative Assay

    The viability test using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for red and yellow Cornus mas (CM) fruit extracts in various ranges of concentration performed after 24 h and 48 h in: ( A ) 3T3-L1 adipocytes, ( B ) SAT-derived human adipocytes (subcutaneous adipose tissue), ( C ) VAT-derived human adipocytes (visceral adipose tissue). DMSO-dimethyl sulfoxide. * p
    Figure Legend Snippet: The viability test using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for red and yellow Cornus mas (CM) fruit extracts in various ranges of concentration performed after 24 h and 48 h in: ( A ) 3T3-L1 adipocytes, ( B ) SAT-derived human adipocytes (subcutaneous adipose tissue), ( C ) VAT-derived human adipocytes (visceral adipose tissue). DMSO-dimethyl sulfoxide. * p

    Techniques Used: MTT Assay, Concentration Assay, Derivative Assay

    Gene expression in 3T3-L1 control adipocytes (C), insulin-resistant adipocytes (IR), and IR adipocytes treated with red and yellow (Yel) CM fruit extracts the concentration of 0.01 mg/mL. * p
    Figure Legend Snippet: Gene expression in 3T3-L1 control adipocytes (C), insulin-resistant adipocytes (IR), and IR adipocytes treated with red and yellow (Yel) CM fruit extracts the concentration of 0.01 mg/mL. * p

    Techniques Used: Expressing, Concentration Assay

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  • 3t3 l1  (ATCC)
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    ATCC 3t3 l1
    Effects of Fa-Trp lipid accumulation in HepG2 cells and <t>3T3-L1</t> adipocytes. ( a ) HepG2 cells were treated with 300 μmol/L palmitic acid conjugated by bovine serum albumin for 24 h. Oil Red O staining of HepG2 cells with Fa-Trp. The number of cells positive for Oil Red O staining was measured by taking a microscopic image of the cell culture and counting the number of cells stained red per 40-mm2 field. ( b ) Mouse 3T3-L1 cells were differentiated into adipocytes. After 24 h of stimulation with Fa-Trp, Oil Red O staining was performed. The relative optical density (OD) values of mature 3T3-L1 adipocytes were determined at OD540. All values are means of three determinations. Errors = s.e. *p
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    Effects of Fa-Trp lipid accumulation in HepG2 cells and 3T3-L1 adipocytes. ( a ) HepG2 cells were treated with 300 μmol/L palmitic acid conjugated by bovine serum albumin for 24 h. Oil Red O staining of HepG2 cells with Fa-Trp. The number of cells positive for Oil Red O staining was measured by taking a microscopic image of the cell culture and counting the number of cells stained red per 40-mm2 field. ( b ) Mouse 3T3-L1 cells were differentiated into adipocytes. After 24 h of stimulation with Fa-Trp, Oil Red O staining was performed. The relative optical density (OD) values of mature 3T3-L1 adipocytes were determined at OD540. All values are means of three determinations. Errors = s.e. *p

    Journal: Scientific Reports

    Article Title: Fatty acid tryptamide from cacao elongates Drosophila melanogaster lifespan with sirtuin-dependent heat shock protein expression

    doi: 10.1038/s41598-022-16471-1

    Figure Lengend Snippet: Effects of Fa-Trp lipid accumulation in HepG2 cells and 3T3-L1 adipocytes. ( a ) HepG2 cells were treated with 300 μmol/L palmitic acid conjugated by bovine serum albumin for 24 h. Oil Red O staining of HepG2 cells with Fa-Trp. The number of cells positive for Oil Red O staining was measured by taking a microscopic image of the cell culture and counting the number of cells stained red per 40-mm2 field. ( b ) Mouse 3T3-L1 cells were differentiated into adipocytes. After 24 h of stimulation with Fa-Trp, Oil Red O staining was performed. The relative optical density (OD) values of mature 3T3-L1 adipocytes were determined at OD540. All values are means of three determinations. Errors = s.e. *p

    Article Snippet: Mouse 3T3-L1 (ATCC CL-173; purchased from ATCC) cells were cultured in DMEM containing 10% fetal bovine serum at 37 °C in a 5% CO2 incubator.

    Techniques: Staining, Cell Culture

    Cytotoxic effect of LM on 3T3-L1 preadipocytes. (a) LM effect on 3T3-L1 preadipocytes viability after 24 h treatment; (b) LM effect on 3T3-L1 preadipocytes viability after 48 h treatment; (c) LM effect on 3T3-L1 preadipocytes viability after 10 days of treatment.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: R-Limonene Enhances Differentiation and 2-Deoxy-D-Glucose Uptake in 3T3-L1 Preadipocytes by Activating the Akt Signaling Pathway

    doi: 10.1155/2018/4573254

    Figure Lengend Snippet: Cytotoxic effect of LM on 3T3-L1 preadipocytes. (a) LM effect on 3T3-L1 preadipocytes viability after 24 h treatment; (b) LM effect on 3T3-L1 preadipocytes viability after 48 h treatment; (c) LM effect on 3T3-L1 preadipocytes viability after 10 days of treatment.

    Article Snippet: Briefly, 3T3-L1 preadipocytes (ATCC, USA) were seeded into 96-well cell culture plates at the density of 1 × 104 cells/well and incubated at 37°C with 5% CO2 for 24 hours.

    Techniques:

    (a)-(b) A competitive study between LM and MK-2206 on Akt phosphorylation at serine 473 during the differentiation of 3T3-L1 preadipocytes. Adipocytes were differentiated in DMI with LM (5 μ M), or MK-2206 (8nM), or LM + MK-2206 individually. LM alone or cotreatment of LM with MK-2206 increased PPAR- γ and Akt activation. MK-2206 alone treated adipocytes exhibited downregulation of PPAR- γ and Akt activation. (a) Western blot analysis of experimental proteins. (b) An optical density of extracted Oil Red O stains from experimental adipocytes. Experiments were performed in triplicate and repeated three times with the same results. Bars display mean ± SEM and statistical analysis was performed by one-way ANOVA. Different letters, A, B, and C, within a column indicate a statistically significant difference ( p

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: R-Limonene Enhances Differentiation and 2-Deoxy-D-Glucose Uptake in 3T3-L1 Preadipocytes by Activating the Akt Signaling Pathway

    doi: 10.1155/2018/4573254

    Figure Lengend Snippet: (a)-(b) A competitive study between LM and MK-2206 on Akt phosphorylation at serine 473 during the differentiation of 3T3-L1 preadipocytes. Adipocytes were differentiated in DMI with LM (5 μ M), or MK-2206 (8nM), or LM + MK-2206 individually. LM alone or cotreatment of LM with MK-2206 increased PPAR- γ and Akt activation. MK-2206 alone treated adipocytes exhibited downregulation of PPAR- γ and Akt activation. (a) Western blot analysis of experimental proteins. (b) An optical density of extracted Oil Red O stains from experimental adipocytes. Experiments were performed in triplicate and repeated three times with the same results. Bars display mean ± SEM and statistical analysis was performed by one-way ANOVA. Different letters, A, B, and C, within a column indicate a statistically significant difference ( p

    Article Snippet: Briefly, 3T3-L1 preadipocytes (ATCC, USA) were seeded into 96-well cell culture plates at the density of 1 × 104 cells/well and incubated at 37°C with 5% CO2 for 24 hours.

    Techniques: Activation Assay, Western Blot

    ODC-AS knockdown and qRT-PCR of marker genes in adipogenesis . 3T3-L1 preadipocytes were infected with lentivirus (Nsi, ODCi-3 or ODCi-5) before the induction for differentiation. Twenty four hours after induction, the cells were collected and examined for mRNA levels of PPARγ, C/EBPα, aP2 and CD36 (see Methods). Data were expressed as means ± SEM (n = 3), relative to Nsi. *compared with Nsi: P

    Journal: Journal of Biomedical Science

    Article Title: Involvement of an alternatively spliced mitochondrial oxodicarboxylate carrier in adipogenesis in 3T3-L1 cells

    doi: 10.1186/1423-0127-16-92

    Figure Lengend Snippet: ODC-AS knockdown and qRT-PCR of marker genes in adipogenesis . 3T3-L1 preadipocytes were infected with lentivirus (Nsi, ODCi-3 or ODCi-5) before the induction for differentiation. Twenty four hours after induction, the cells were collected and examined for mRNA levels of PPARγ, C/EBPα, aP2 and CD36 (see Methods). Data were expressed as means ± SEM (n = 3), relative to Nsi. *compared with Nsi: P

    Article Snippet: 3T3-L1 preadipocytes (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Invitrogen).

    Techniques: Quantitative RT-PCR, Marker, Infection

    Quantitation of GLUT4 translocation and glucose uptake in 3T3-L1 cells. (A) TIRF microscopy based quantitation of GLUT4 translocation in 3T3-L1 GLUT4-GFP adipocytes. Cells were grown in 96-well plates (20,000 cells/well) for 24 hours and then starved overnight in serum-free medium. Images before and 10 minutes after stimulation with indicated substances are shown. Scale bar = 20 μm. (B) GLUT4-GFP signal intensity increase was analyzed after 10 and 30 minutes of stimulation. Error bars are based on the standard error of the mean. **P

    Journal: PLoS ONE

    Article Title: Biomolecular Characterization of Putative Antidiabetic Herbal Extracts

    doi: 10.1371/journal.pone.0148109

    Figure Lengend Snippet: Quantitation of GLUT4 translocation and glucose uptake in 3T3-L1 cells. (A) TIRF microscopy based quantitation of GLUT4 translocation in 3T3-L1 GLUT4-GFP adipocytes. Cells were grown in 96-well plates (20,000 cells/well) for 24 hours and then starved overnight in serum-free medium. Images before and 10 minutes after stimulation with indicated substances are shown. Scale bar = 20 μm. (B) GLUT4-GFP signal intensity increase was analyzed after 10 and 30 minutes of stimulation. Error bars are based on the standard error of the mean. **P

    Article Snippet: 3T3-L1 cells were purchased from ATCC (Manassas, USA).

    Techniques: Quantitation Assay, Translocation Assay, Microscopy