3t3 l1 cells  (ATCC)


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    ATCC 3t3 l1 cells
    DEX induces the change in NCoR mRNA splicing observed during <t>3T3-L1</t> cells differentiation. Cells were differentiated with various combinations of the individual factors within the tripartite differentiation cocktail. Means ± S.E.M. (n = 3 for SMRT Exon 40 and NCoR Exon 28, and n = 5 for NCoR Exon 37) are presented. p
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    1) Product Images from "Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling"

    Article Title: Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling

    Journal: Molecular and cellular endocrinology

    doi: 10.1016/j.mce.2015.06.036

    DEX induces the change in NCoR mRNA splicing observed during 3T3-L1 cells differentiation. Cells were differentiated with various combinations of the individual factors within the tripartite differentiation cocktail. Means ± S.E.M. (n = 3 for SMRT Exon 40 and NCoR Exon 28, and n = 5 for NCoR Exon 37) are presented. p
    Figure Legend Snippet: DEX induces the change in NCoR mRNA splicing observed during 3T3-L1 cells differentiation. Cells were differentiated with various combinations of the individual factors within the tripartite differentiation cocktail. Means ± S.E.M. (n = 3 for SMRT Exon 40 and NCoR Exon 28, and n = 5 for NCoR Exon 37) are presented. p

    Techniques Used:

    2) Product Images from "Inula japonica Thunb. Flower Ethanol Extract Improves Obesity and Exercise Endurance in Mice Fed a High-Fat Diet"

    Article Title: Inula japonica Thunb. Flower Ethanol Extract Improves Obesity and Exercise Endurance in Mice Fed a High-Fat Diet

    Journal: Nutrients

    doi: 10.3390/nu11010017

    Effects of I. japonica flower ethanol extract (IJE) on adipogenic differentiation of 3T3-L1 cells. ( A ) The effects of IJE on cell viability were measured by the MTT assay. ( B ) Oil Red O staining of differentiated 3T3-L1 cells with or without IJE treatment was done (scale bar: 100 μm), after which absorbance was measured at 500 nm in triplicate. ( C ) Optical density (OD). ( D ) Relative mRNA and ( E ) protein levels of PPARγ, C/EBPα, FAS, and aP2. Data are presented mean ± standard deviation (SD) of triplicate experiments. #, ##, and ### indicate p
    Figure Legend Snippet: Effects of I. japonica flower ethanol extract (IJE) on adipogenic differentiation of 3T3-L1 cells. ( A ) The effects of IJE on cell viability were measured by the MTT assay. ( B ) Oil Red O staining of differentiated 3T3-L1 cells with or without IJE treatment was done (scale bar: 100 μm), after which absorbance was measured at 500 nm in triplicate. ( C ) Optical density (OD). ( D ) Relative mRNA and ( E ) protein levels of PPARγ, C/EBPα, FAS, and aP2. Data are presented mean ± standard deviation (SD) of triplicate experiments. #, ##, and ### indicate p

    Techniques Used: MTT Assay, Staining, Standard Deviation

    3) Product Images from "Lipoatrophy and metabolic disturbance in mice with adipose-specific deletion of kindlin-2"

    Article Title: Lipoatrophy and metabolic disturbance in mice with adipose-specific deletion of kindlin-2

    Journal: JCI Insight

    doi: 10.1172/jci.insight.128405

    Deletion of kindlin-2 inhibits adipogenesis in vitro. ( A ) Western blot analysis of protein levels of adipogenesis genes in iWAT of WT and KO mice. ( B ) Oil Red O staining of 3T3-L1 cells treated with control siRNA or kindlin-2 siRNA (si-con and si-K2, respectively) after differentiation ( N = 3 replicates). Original magnification, ×200 (top) and ×100 (bottom). ( C ) Quantitative real-time reverse transcriptase PCR (qPCR) analyses of mRNA levels of adipogenesis genes in si-con and si-K2 cells ( N = 3 replicates). ( D ) Western blot analysis of adipogenesis protein expression in si-con and si-K2 cells ( N = 3 replicates). ( E ) Oil Red O staining of WT and KO stromal vessel fraction (SVF) cells after differentiation. Original magnification, ×200. * P
    Figure Legend Snippet: Deletion of kindlin-2 inhibits adipogenesis in vitro. ( A ) Western blot analysis of protein levels of adipogenesis genes in iWAT of WT and KO mice. ( B ) Oil Red O staining of 3T3-L1 cells treated with control siRNA or kindlin-2 siRNA (si-con and si-K2, respectively) after differentiation ( N = 3 replicates). Original magnification, ×200 (top) and ×100 (bottom). ( C ) Quantitative real-time reverse transcriptase PCR (qPCR) analyses of mRNA levels of adipogenesis genes in si-con and si-K2 cells ( N = 3 replicates). ( D ) Western blot analysis of adipogenesis protein expression in si-con and si-K2 cells ( N = 3 replicates). ( E ) Oil Red O staining of WT and KO stromal vessel fraction (SVF) cells after differentiation. Original magnification, ×200. * P

    Techniques Used: In Vitro, Western Blot, Mouse Assay, Staining, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing

    4) Product Images from "Anandamide-derived Prostamide F2α"

    Article Title: Anandamide-derived Prostamide F2α

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.489906

    Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α
    Figure Legend Snippet: Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ
    Figure Legend Snippet: Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ

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

    Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ
    Figure Legend Snippet: Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ

    Techniques Used: Inhibition, Activity Assay, Real-time Polymerase Chain Reaction

    Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence
    Figure Legend Snippet: Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence

    Techniques Used: Mass Spectrometry

    5) Product Images from "Anandamide-derived Prostamide F2α"

    Article Title: Anandamide-derived Prostamide F2α

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.489906

    Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α
    Figure Legend Snippet: Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ
    Figure Legend Snippet: Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ

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

    Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ
    Figure Legend Snippet: Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ

    Techniques Used: Inhibition, Activity Assay, Real-time Polymerase Chain Reaction

    Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence
    Figure Legend Snippet: Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence

    Techniques Used: Mass Spectrometry

    6) Product Images from "Anandamide-derived Prostamide F2α"

    Article Title: Anandamide-derived Prostamide F2α

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.489906

    Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α
    Figure Legend Snippet: Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ
    Figure Legend Snippet: Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ

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

    Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ
    Figure Legend Snippet: Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ

    Techniques Used: Inhibition, Activity Assay, Real-time Polymerase Chain Reaction

    Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence
    Figure Legend Snippet: Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence

    Techniques Used: Mass Spectrometry

    7) Product Images from "Biomolecular Characterization of Putative Antidiabetic Herbal Extracts"

    Article Title: Biomolecular Characterization of Putative Antidiabetic Herbal Extracts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0148109

    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
    Figure Legend 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

    Techniques Used: Quantitation Assay, Translocation Assay, Microscopy

    8) Product Images from "Myonectin inhibits adipogenesis in 3T3-L1 preadipocytes by regulating p38 MAPK pathway"

    Article Title: Myonectin inhibits adipogenesis in 3T3-L1 preadipocytes by regulating p38 MAPK pathway

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2021.54.2.262

    Suppression of adipogenic differentiation by treatment with myonectin. (A) Effects of myonectin on cell viability. The viability of untreated control cells (Non) was defined as 100%. (B) Effects of myonectin on the differentiation of 3T3-L1 cells. Differentiation of con-fluent 3T3-L1 cells was induced by the MDI cocktail (0.5 mM IBMX, 1 μM Dexamethasone, and 10 μg/ml insulin). The 3T3-L1 cells were treated with different concentrations (0.05-2 μg/ml) of myonectin during differentiation. On day 10 of differentiation, the fully differentiated adipocytes were fixed and stained with oil red O solution for visualization of the lipid accumulation. (C-F) mRNA levels of adipogenic genes in mature 3T3-L1 adipocytes. Each gene was normalized to Rpl32. All quantitative data are the means ± SD (n = 6). *P
    Figure Legend Snippet: Suppression of adipogenic differentiation by treatment with myonectin. (A) Effects of myonectin on cell viability. The viability of untreated control cells (Non) was defined as 100%. (B) Effects of myonectin on the differentiation of 3T3-L1 cells. Differentiation of con-fluent 3T3-L1 cells was induced by the MDI cocktail (0.5 mM IBMX, 1 μM Dexamethasone, and 10 μg/ml insulin). The 3T3-L1 cells were treated with different concentrations (0.05-2 μg/ml) of myonectin during differentiation. On day 10 of differentiation, the fully differentiated adipocytes were fixed and stained with oil red O solution for visualization of the lipid accumulation. (C-F) mRNA levels of adipogenic genes in mature 3T3-L1 adipocytes. Each gene was normalized to Rpl32. All quantitative data are the means ± SD (n = 6). *P

    Techniques Used: Staining

    9) Product Images from "Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling"

    Article Title: Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling

    Journal: Scientific Reports

    doi: 10.1038/srep42104

    Rosiglitazone Induces Varied Adipogenic Activities Based on Cell Culture Plastic. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) using various tissue culture plates. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone cultured in Greiner Bio-One CELLSTAR™, Brandtech cellGrade™, and Labnet International Krystal™ 2000 tissue culture plates for ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for rosiglitazone cultured in the tissue culture plates described above for ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for rosiglitazone cultured in the tissue culture plates described above for ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Fold induction of triglyceride accumulative response over vehicle control for rosiglitazone cultured in the cell culture plastics described above for ATCC 3T3-L1 cells ( J ), Zenbio 3T3-L1 cells ( K ), and OP9 cells ( L ). Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p
    Figure Legend Snippet: Rosiglitazone Induces Varied Adipogenic Activities Based on Cell Culture Plastic. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) using various tissue culture plates. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone cultured in Greiner Bio-One CELLSTAR™, Brandtech cellGrade™, and Labnet International Krystal™ 2000 tissue culture plates for ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for rosiglitazone cultured in the tissue culture plates described above for ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for rosiglitazone cultured in the tissue culture plates described above for ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Fold induction of triglyceride accumulative response over vehicle control for rosiglitazone cultured in the cell culture plastics described above for ATCC 3T3-L1 cells ( J ), Zenbio 3T3-L1 cells ( K ), and OP9 cells ( L ). Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p

    Techniques Used: Cell Culture, Staining, Concentration Assay

    Bisphenol A and Analogs Induce Varied Adipogenic Activities Between Cell Lines. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with four bisphenol A analogs. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for bisphenol A (BPA), tetrabrominated bisphenol A (TBBPA), tetrachlorinated bisphenol A (TCBPA), and hexafluorinated bisphenol A (BPAF) in ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Triglyceride accumulation responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p
    Figure Legend Snippet: Bisphenol A and Analogs Induce Varied Adipogenic Activities Between Cell Lines. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with four bisphenol A analogs. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for bisphenol A (BPA), tetrabrominated bisphenol A (TBBPA), tetrachlorinated bisphenol A (TCBPA), and hexafluorinated bisphenol A (BPAF) in ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Triglyceride accumulation responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p

    Techniques Used: Staining, Activity Assay, Concentration Assay

    Mechanistic Receptor Controls Induce Varied Adipogenic Activities Between Cell Lines. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with mechanistic receptor control ligands. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone (RSG, PPARγ agonist), GW3965 (liver X receptor, LXR, agonist), dexamethasone (glucocorticoid receptor, GR, agonist), 1–850 (thyroid receptor, TR, antagonist), flutamide (androgen receptor, AR, antagonist), and LG100268 (retinoid X receptor, RXR, agonist) in ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p
    Figure Legend Snippet: Mechanistic Receptor Controls Induce Varied Adipogenic Activities Between Cell Lines. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with mechanistic receptor control ligands. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone (RSG, PPARγ agonist), GW3965 (liver X receptor, LXR, agonist), dexamethasone (glucocorticoid receptor, GR, agonist), 1–850 (thyroid receptor, TR, antagonist), flutamide (androgen receptor, AR, antagonist), and LG100268 (retinoid X receptor, RXR, agonist) in ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p

    Techniques Used: Staining, Activity Assay, Concentration Assay

    10) Product Images from "Adipogenesis is differentially impaired by thyroid hormone receptor mutant isoforms"

    Article Title: Adipogenesis is differentially impaired by thyroid hormone receptor mutant isoforms

    Journal: Journal of molecular endocrinology

    doi: 10.1677/JME-09-0137

    )). Lane 3 indicates control cells that were used as negative controls, indicating the specific bands detected in lanes 1 and 2. GAPDH was used as a loading control. (B) Total cellular lysates (30 μg) were used in the western blot analysis. Endogenous TRα1 and TRβ1 receptor proteins in L1-α1PV cells (lane 1), L1-β1PV cells (lane 2), and 3T3-L1 cells (lane 3) were detected by monoclonal antibody C4 that recognizes the C-terminus ofTRβ1 and TRα1 receptors. GAPDH wasusedasa loading control.
    Figure Legend Snippet: )). Lane 3 indicates control cells that were used as negative controls, indicating the specific bands detected in lanes 1 and 2. GAPDH was used as a loading control. (B) Total cellular lysates (30 μg) were used in the western blot analysis. Endogenous TRα1 and TRβ1 receptor proteins in L1-α1PV cells (lane 1), L1-β1PV cells (lane 2), and 3T3-L1 cells (lane 3) were detected by monoclonal antibody C4 that recognizes the C-terminus ofTRβ1 and TRα1 receptors. GAPDH wasusedasa loading control.

    Techniques Used: Western Blot

    T 3 -induced adipogenesis in 3T3-L1 cells is differentially impaired by TR mutant isoforms. Control 3T3-L1, L1-β1PV, and L1-α1PV cells were induced to undergo adipogenesis in the absence (panels 1, 2, and 3) or presence of T 3 (2 nM, panels 4, 5, and 6) as described in Materials and methods. After 7 days, matured adipocytes with lipid droplets were visualized by staining with Oil Red O (A) and by phase contrast microscopy (C). (B) Quantification of relative Oil Red O staining intensities of lipid droplets in matured adipocytes. Data are expressed as mean±S.E.M. ( n =6). The P values are indicated.
    Figure Legend Snippet: T 3 -induced adipogenesis in 3T3-L1 cells is differentially impaired by TR mutant isoforms. Control 3T3-L1, L1-β1PV, and L1-α1PV cells were induced to undergo adipogenesis in the absence (panels 1, 2, and 3) or presence of T 3 (2 nM, panels 4, 5, and 6) as described in Materials and methods. After 7 days, matured adipocytes with lipid droplets were visualized by staining with Oil Red O (A) and by phase contrast microscopy (C). (B) Quantification of relative Oil Red O staining intensities of lipid droplets in matured adipocytes. Data are expressed as mean±S.E.M. ( n =6). The P values are indicated.

    Techniques Used: Mutagenesis, Staining, Microscopy

    11) Product Images from "Flightless-I Controls Fat Storage in Drosophila"

    Article Title: Flightless-I Controls Fat Storage in Drosophila

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2018.0120

    Drosophila fliI inhibits lipid storage by suppressing mRNA expression of desaturase 1 (A) Quantitative RT-PCR analysis of desat1 in the intestine (left) and fat body (right) from 7-day-old dissected adults. mRNA expression of desat1 was higher in fliI 3/14 mutants relative to w 1118 flies (controls). (B) The mRNA expression of desat1 gradually increased in w 1118 flies (control) and fliI 3/14 mutants. The transcript levels of desat1 at the indicated time points were significantly higher in fliI 3/14 mutants than in w 1118 flies. (C) For 10 days of RU486 supplementation, the levels of triglycerides in group “P{Switch1}106 > fliI, +RU486” significantly decreased relative to the RU486 control (P{Switch1}106 > fliI, no RU486). (D) Quantitative RT-PCR analysis of desat1 expression (normalized to Rp49 mRNA levels) in whole-body extracts of flies reared under conditions identical to those in (C). The overexpression of fliI under the influence of RU486 supplementation suppressed the mRNA expression of desat1 . (E) The analysis of mRNA expression in empty-vector–transfected cells or in FliI-expressing 3T3-L1 cells. The overexpression of FliI-downregulated transcripts of murine stearoyl-CoA desaturase genes, such as SCD1 , SCD2 , and SCD4 . (F) Overexpression of fliI (Act5C GS > fliI, +RU486) significantly increased the sensitivity to starvation as compared to RU486 control (Act5C GS > fliI, no RU486) according to the logrank test ( P
    Figure Legend Snippet: Drosophila fliI inhibits lipid storage by suppressing mRNA expression of desaturase 1 (A) Quantitative RT-PCR analysis of desat1 in the intestine (left) and fat body (right) from 7-day-old dissected adults. mRNA expression of desat1 was higher in fliI 3/14 mutants relative to w 1118 flies (controls). (B) The mRNA expression of desat1 gradually increased in w 1118 flies (control) and fliI 3/14 mutants. The transcript levels of desat1 at the indicated time points were significantly higher in fliI 3/14 mutants than in w 1118 flies. (C) For 10 days of RU486 supplementation, the levels of triglycerides in group “P{Switch1}106 > fliI, +RU486” significantly decreased relative to the RU486 control (P{Switch1}106 > fliI, no RU486). (D) Quantitative RT-PCR analysis of desat1 expression (normalized to Rp49 mRNA levels) in whole-body extracts of flies reared under conditions identical to those in (C). The overexpression of fliI under the influence of RU486 supplementation suppressed the mRNA expression of desat1 . (E) The analysis of mRNA expression in empty-vector–transfected cells or in FliI-expressing 3T3-L1 cells. The overexpression of FliI-downregulated transcripts of murine stearoyl-CoA desaturase genes, such as SCD1 , SCD2 , and SCD4 . (F) Overexpression of fliI (Act5C GS > fliI, +RU486) significantly increased the sensitivity to starvation as compared to RU486 control (Act5C GS > fliI, no RU486) according to the logrank test ( P

    Techniques Used: Expressing, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Transfection

    12) Product Images from "Fatty Acid 2-Hydroxylase Mediates Diffusional Mobility of Raft-associated Lipids, GLUT4 Level, and Lipogenesis in 3T3-L1 Adipocytes *"

    Article Title: Fatty Acid 2-Hydroxylase Mediates Diffusional Mobility of Raft-associated Lipids, GLUT4 Level, and Lipogenesis in 3T3-L1 Adipocytes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.119933

    Treatment of siRNAs against FA2H blocks hormone-induced differentiation of 3T3-L1 adipocytes. A ). B , mRNA samples from differentiating 3T3-L1 cells at the indicated stages were prepared, and the FA2H levels were analyzed by RT-PCR as described under “Experimental Procedures.” C , 3T3-L1 cells were treated with a negative control ( NC ) siRNA or siRNAs recognizing FA2H (sequence ( seq ) 1 and sequence 2), and mRNA samples were prepared 2 days after transfection. FA2H levels were analyzed by RT-PCR. D and E , 3T3-L1 cells pretreated with a negative control siRNA or siRNAs recognizing FA2H (sequence 1 and sequence 2) were induced to differentiation. On day 8, mRNA samples were prepared, and the Pref1, peroxisome proliferator-activated receptor γ, SCD1, and CD36 levels were analyzed by RT-PCR. The results represent means ± S.E. of three independent experiments. *, p
    Figure Legend Snippet: Treatment of siRNAs against FA2H blocks hormone-induced differentiation of 3T3-L1 adipocytes. A ). B , mRNA samples from differentiating 3T3-L1 cells at the indicated stages were prepared, and the FA2H levels were analyzed by RT-PCR as described under “Experimental Procedures.” C , 3T3-L1 cells were treated with a negative control ( NC ) siRNA or siRNAs recognizing FA2H (sequence ( seq ) 1 and sequence 2), and mRNA samples were prepared 2 days after transfection. FA2H levels were analyzed by RT-PCR. D and E , 3T3-L1 cells pretreated with a negative control siRNA or siRNAs recognizing FA2H (sequence 1 and sequence 2) were induced to differentiation. On day 8, mRNA samples were prepared, and the Pref1, peroxisome proliferator-activated receptor γ, SCD1, and CD36 levels were analyzed by RT-PCR. The results represent means ± S.E. of three independent experiments. *, p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Negative Control, Sequencing, Transfection

    Treatment of siRNAs against FA2H blocks TAG accumulation during differentiation of 3T3-L1 adipocytes. A , 3T3-L1 cells pretreated with a negative control ( NC ) siRNA or siRNAs recognizing FA2H (sequence ( seq ) 1 and sequence 2) were induced to differentiation. On day 8, total lipids were extracted, and TAG were measured by quantitative gas chromatography and normalized to total protein as described under “Experimental Procedures.” Results are the means ± S.E. of three independent experiments. *, p
    Figure Legend Snippet: Treatment of siRNAs against FA2H blocks TAG accumulation during differentiation of 3T3-L1 adipocytes. A , 3T3-L1 cells pretreated with a negative control ( NC ) siRNA or siRNAs recognizing FA2H (sequence ( seq ) 1 and sequence 2) were induced to differentiation. On day 8, total lipids were extracted, and TAG were measured by quantitative gas chromatography and normalized to total protein as described under “Experimental Procedures.” Results are the means ± S.E. of three independent experiments. *, p

    Techniques Used: Negative Control, Sequencing, Gas Chromatography

    13) Product Images from "Fatty Acid 2-Hydroxylase Mediates Diffusional Mobility of Raft-associated Lipids, GLUT4 Level, and Lipogenesis in 3T3-L1 Adipocytes *"

    Article Title: Fatty Acid 2-Hydroxylase Mediates Diffusional Mobility of Raft-associated Lipids, GLUT4 Level, and Lipogenesis in 3T3-L1 Adipocytes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.119933

    Treatment of siRNAs against FA2H blocks hormone-induced differentiation of 3T3-L1 adipocytes. A ). B , mRNA samples from differentiating 3T3-L1 cells at the indicated stages were prepared, and the FA2H levels were analyzed by RT-PCR as described under “Experimental Procedures.” C , 3T3-L1 cells were treated with a negative control ( NC ) siRNA or siRNAs recognizing FA2H (sequence ( seq ) 1 and sequence 2), and mRNA samples were prepared 2 days after transfection. FA2H levels were analyzed by RT-PCR. D and E , 3T3-L1 cells pretreated with a negative control siRNA or siRNAs recognizing FA2H (sequence 1 and sequence 2) were induced to differentiation. On day 8, mRNA samples were prepared, and the Pref1, peroxisome proliferator-activated receptor γ, SCD1, and CD36 levels were analyzed by RT-PCR. The results represent means ± S.E. of three independent experiments. *, p
    Figure Legend Snippet: Treatment of siRNAs against FA2H blocks hormone-induced differentiation of 3T3-L1 adipocytes. A ). B , mRNA samples from differentiating 3T3-L1 cells at the indicated stages were prepared, and the FA2H levels were analyzed by RT-PCR as described under “Experimental Procedures.” C , 3T3-L1 cells were treated with a negative control ( NC ) siRNA or siRNAs recognizing FA2H (sequence ( seq ) 1 and sequence 2), and mRNA samples were prepared 2 days after transfection. FA2H levels were analyzed by RT-PCR. D and E , 3T3-L1 cells pretreated with a negative control siRNA or siRNAs recognizing FA2H (sequence 1 and sequence 2) were induced to differentiation. On day 8, mRNA samples were prepared, and the Pref1, peroxisome proliferator-activated receptor γ, SCD1, and CD36 levels were analyzed by RT-PCR. The results represent means ± S.E. of three independent experiments. *, p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Negative Control, Sequencing, Transfection

    Treatment of siRNAs against FA2H blocks TAG accumulation during differentiation of 3T3-L1 adipocytes. A , 3T3-L1 cells pretreated with a negative control ( NC ) siRNA or siRNAs recognizing FA2H (sequence ( seq ) 1 and sequence 2) were induced to differentiation. On day 8, total lipids were extracted, and TAG were measured by quantitative gas chromatography and normalized to total protein as described under “Experimental Procedures.” Results are the means ± S.E. of three independent experiments. *, p
    Figure Legend Snippet: Treatment of siRNAs against FA2H blocks TAG accumulation during differentiation of 3T3-L1 adipocytes. A , 3T3-L1 cells pretreated with a negative control ( NC ) siRNA or siRNAs recognizing FA2H (sequence ( seq ) 1 and sequence 2) were induced to differentiation. On day 8, total lipids were extracted, and TAG were measured by quantitative gas chromatography and normalized to total protein as described under “Experimental Procedures.” Results are the means ± S.E. of three independent experiments. *, p

    Techniques Used: Negative Control, Sequencing, Gas Chromatography

    14) Product Images from "Ginger (Zingiber officinale) Attenuates Obesity and Adipose Tissue Remodeling in High-Fat Diet-Fed C57BL/6 Mice"

    Article Title: Ginger (Zingiber officinale) Attenuates Obesity and Adipose Tissue Remodeling in High-Fat Diet-Fed C57BL/6 Mice

    Journal: International Journal of Environmental Research and Public Health

    doi: 10.3390/ijerph18020631

    Ginger reduced lipid accumulation in 3T3-L1 adipocytes. ( A ) Total polyphenol and flavonoid contents of ginger extract; the culture of 3T3-L1 cells was incubated with ginger extract at different doses (15–120 μg/mL). 2,3-Bis-(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) reagent was added for 3 h into 96-well plates to measure cell viability. ( B ) Cell viability by XTT; the 3T3-L1 cells were differentiated in the presence or absence of ginger extract (0–60 μg/mL) for 7 days. Dimethyl sulfoxide (DMSO) was used as a vehicle control. ( C ) Triglyceride (TG) accumulation in 3T3-L1 adipocytes visualized using Oil Red O (ORO) staining. Representative images from three separate experiments are shown (20× magnification, upper). Images were cropped to the relevant part of the field without altering the resolution (close-cropped images, lower). ( D ) ORO dye was extracted using isopropanol and quantified as the optical density at 500 nm (OD 500 ). ( E ) Heme oxygenase 1 ( HO-1 ) gene expression determined using real-time PCR. Data are represented as the mean ± standard error of the mean (SEM) of three independent experiments. * p
    Figure Legend Snippet: Ginger reduced lipid accumulation in 3T3-L1 adipocytes. ( A ) Total polyphenol and flavonoid contents of ginger extract; the culture of 3T3-L1 cells was incubated with ginger extract at different doses (15–120 μg/mL). 2,3-Bis-(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) reagent was added for 3 h into 96-well plates to measure cell viability. ( B ) Cell viability by XTT; the 3T3-L1 cells were differentiated in the presence or absence of ginger extract (0–60 μg/mL) for 7 days. Dimethyl sulfoxide (DMSO) was used as a vehicle control. ( C ) Triglyceride (TG) accumulation in 3T3-L1 adipocytes visualized using Oil Red O (ORO) staining. Representative images from three separate experiments are shown (20× magnification, upper). Images were cropped to the relevant part of the field without altering the resolution (close-cropped images, lower). ( D ) ORO dye was extracted using isopropanol and quantified as the optical density at 500 nm (OD 500 ). ( E ) Heme oxygenase 1 ( HO-1 ) gene expression determined using real-time PCR. Data are represented as the mean ± standard error of the mean (SEM) of three independent experiments. * p

    Techniques Used: Incubation, Staining, Expressing, Real-time Polymerase Chain Reaction

    15) Product Images from "The orphan ligand, Activin C, signals through activin receptor-like kinase 7"

    Article Title: The orphan ligand, Activin C, signals through activin receptor-like kinase 7

    Journal: bioRxiv

    doi: 10.1101/2022.03.16.484571

    ActC activates SMAD2 through ALK7 in differentiated adipocytes. ( A ) Representative images of isolated adipose-derived stromal vascular fraction (SVF) or cultured 3T3-L1 cells prior to differentiation ( left , Day 0) and following differentiation ( right , Day 10). Scale bars are 20 μm. Schematic shown in upper right for visualization of timeline. ( B ) Western blot (WB) showing phosphorylated SMAD2 (pSMAD2) and total SMAD2/3 in 3T3-L1-derived adipocytes following treatment with ActA, ActB, or ActC (2 nM) for 1h. ( C ) WB following treatment of SVF-derived adipocytes with ActB or ActC (2 nM) with or without Fst288 (800 ng/ml) for 1h. ( D ) Quantitative PCR (RT-qPCR) of Acvr1c expression in differentiated 3T3-L1 cells and SVF adipocytes. Bars display mean ± SD of three experimental replicates. ( E ) WB following treatment of SVF-derived adipocytes with ActB or ActC (2 nM) in the presence or absence of a neutralizing antibody targeting ALK7 for 1h. ( F ) Representative images of SVF-derived adipocytes following treatment with ActA, ActB, or ActC during differentiation with or without Fst288. ( G ) RT-qPCR of target genes Pparg2, Cebpa , and Pnpla2 following treatment with ActA, ActB, or ActC during differentiation. Oil Red O quantification based on images in ( F ). Significance is represented as: * p
    Figure Legend Snippet: ActC activates SMAD2 through ALK7 in differentiated adipocytes. ( A ) Representative images of isolated adipose-derived stromal vascular fraction (SVF) or cultured 3T3-L1 cells prior to differentiation ( left , Day 0) and following differentiation ( right , Day 10). Scale bars are 20 μm. Schematic shown in upper right for visualization of timeline. ( B ) Western blot (WB) showing phosphorylated SMAD2 (pSMAD2) and total SMAD2/3 in 3T3-L1-derived adipocytes following treatment with ActA, ActB, or ActC (2 nM) for 1h. ( C ) WB following treatment of SVF-derived adipocytes with ActB or ActC (2 nM) with or without Fst288 (800 ng/ml) for 1h. ( D ) Quantitative PCR (RT-qPCR) of Acvr1c expression in differentiated 3T3-L1 cells and SVF adipocytes. Bars display mean ± SD of three experimental replicates. ( E ) WB following treatment of SVF-derived adipocytes with ActB or ActC (2 nM) in the presence or absence of a neutralizing antibody targeting ALK7 for 1h. ( F ) Representative images of SVF-derived adipocytes following treatment with ActA, ActB, or ActC during differentiation with or without Fst288. ( G ) RT-qPCR of target genes Pparg2, Cebpa , and Pnpla2 following treatment with ActA, ActB, or ActC during differentiation. Oil Red O quantification based on images in ( F ). Significance is represented as: * p

    Techniques Used: Isolation, Derivative Assay, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    16) Product Images from "SINO Syndrome Causative KIDINS220/ARMS Gene Regulates Adipocyte Differentiation"

    Article Title: SINO Syndrome Causative KIDINS220/ARMS Gene Regulates Adipocyte Differentiation

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.619475

    KIDINS220/ARMS inhibited adipocyte differentiation. The mRNA (A) and protein (B) levels of KIDINS220/ARMS are gradually decreased along with the differentiation process of preadipocyte 3T3-L1 cells. The overexpression (C) and knockdown (D) of KINDIS220/ARMS in 3T3-L1 cells were confirmed by quantitative PCR and immunoblotting. (E) 3T3-L1 preadipocytes were transfected with KIDINS220-Flag or KIDINS220 siRNA, respectively, and then induced to differentiation 48 h later (Day 0). On Day 8, cytoplasmic triacylglycerol was stained with Oil Red O. (F) The densities of staining were calculated by the absorbance at 490 nm. Student’s unpaired t -test; ** p
    Figure Legend Snippet: KIDINS220/ARMS inhibited adipocyte differentiation. The mRNA (A) and protein (B) levels of KIDINS220/ARMS are gradually decreased along with the differentiation process of preadipocyte 3T3-L1 cells. The overexpression (C) and knockdown (D) of KINDIS220/ARMS in 3T3-L1 cells were confirmed by quantitative PCR and immunoblotting. (E) 3T3-L1 preadipocytes were transfected with KIDINS220-Flag or KIDINS220 siRNA, respectively, and then induced to differentiation 48 h later (Day 0). On Day 8, cytoplasmic triacylglycerol was stained with Oil Red O. (F) The densities of staining were calculated by the absorbance at 490 nm. Student’s unpaired t -test; ** p

    Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Transfection, Staining

    ERK pathway is required for KIDINS220/ARMS mediated adipocyte maturation. (A) 3T3-L1 cells were induced to differentiation, and cells were harvested for western blot analysis on Days 0, 2, and 4. (B,C) 3T3-L1 cells were transfected with KIDINS220-Flag (B) or KIDINS220 siRNA (C) and cultured for 2 days. Then, cells were induced to differentiation (Day 0), and ERK inhibitor (Ravoxertinib) (B) or agonist (Honokiol) (C) were added to the medium at Day 1, respectively. On Days 0, 2, and 4, cells were harvested and analyzed by western blot for the phosphorylation of ERK. (D) Images of Oil-red O staining performed 8 days after the differentiation. Ravoxertinib and Honokiol: cells treated with 1 mM Ravoxertinib or 10 mM Honokiol alone; KIDINS220-Flag and KIDINS220 siRNA: cells were transfected with KIDINS220-Flag or KIDINS220 siRNA, respectively; KIDINS220-Flag + Ravoxertinib: cells were transfected with KIDINS220-Flag and treated with 1 mM Ravoxertinib; KIDINS220 siRNA + Honokiol: cells were transfected with KIDINS220 siRNA and treated with 10 mM Honokiol. (E) The densities of staining were calculated by the absorbance at 490 nm. Student’s unpaired t -test; * p
    Figure Legend Snippet: ERK pathway is required for KIDINS220/ARMS mediated adipocyte maturation. (A) 3T3-L1 cells were induced to differentiation, and cells were harvested for western blot analysis on Days 0, 2, and 4. (B,C) 3T3-L1 cells were transfected with KIDINS220-Flag (B) or KIDINS220 siRNA (C) and cultured for 2 days. Then, cells were induced to differentiation (Day 0), and ERK inhibitor (Ravoxertinib) (B) or agonist (Honokiol) (C) were added to the medium at Day 1, respectively. On Days 0, 2, and 4, cells were harvested and analyzed by western blot for the phosphorylation of ERK. (D) Images of Oil-red O staining performed 8 days after the differentiation. Ravoxertinib and Honokiol: cells treated with 1 mM Ravoxertinib or 10 mM Honokiol alone; KIDINS220-Flag and KIDINS220 siRNA: cells were transfected with KIDINS220-Flag or KIDINS220 siRNA, respectively; KIDINS220-Flag + Ravoxertinib: cells were transfected with KIDINS220-Flag and treated with 1 mM Ravoxertinib; KIDINS220 siRNA + Honokiol: cells were transfected with KIDINS220 siRNA and treated with 10 mM Honokiol. (E) The densities of staining were calculated by the absorbance at 490 nm. Student’s unpaired t -test; * p

    Techniques Used: Western Blot, Transfection, Cell Culture, Staining

    17) Product Images from "A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation"

    Article Title: A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation

    Journal: Nature cell biology

    doi: 10.1038/35060059

    PKal is present during adipogenesis and is required for differentiation of 3T3-L1 cells a , b , Casein/Plg zymograms. a , Incubation in substrate buffer. FBS, fetal bovine serum; CM, conditioned medium; MG, mammary-gland lysate; NHP, normal human plasma; MS, mouse serum. b , Incubation in substrate buffer alone or in buffer containing WT ecotin (EcoWT) or EcoRR. Inhibited bands are indicated by arrowheads. Human enzymes are given the prefix ‘h’. Pls, plasmin. c , Western blotting to detect PKal. PKal– HS, prekallikrein-deficient human plasma. d , e , Staining with Oil red O of preadipocytes grown in normal ( d ) or prekallikrein-deficient ( e ) human plasma. f–i , Staining with Oil red O of adipocytes differentiated in FBS ( f ), normal human plasma ( g ), prekallikrein-deficient human plasma ( h ), or prekallikrein-deficient human plasma with exogenous human PKal added ( i ). j , Quantification of adipose conversion; absorbance is designated as 1.0 for cells grown in NHP. Data are means ± s.d.
    Figure Legend Snippet: PKal is present during adipogenesis and is required for differentiation of 3T3-L1 cells a , b , Casein/Plg zymograms. a , Incubation in substrate buffer. FBS, fetal bovine serum; CM, conditioned medium; MG, mammary-gland lysate; NHP, normal human plasma; MS, mouse serum. b , Incubation in substrate buffer alone or in buffer containing WT ecotin (EcoWT) or EcoRR. Inhibited bands are indicated by arrowheads. Human enzymes are given the prefix ‘h’. Pls, plasmin. c , Western blotting to detect PKal. PKal– HS, prekallikrein-deficient human plasma. d , e , Staining with Oil red O of preadipocytes grown in normal ( d ) or prekallikrein-deficient ( e ) human plasma. f–i , Staining with Oil red O of adipocytes differentiated in FBS ( f ), normal human plasma ( g ), prekallikrein-deficient human plasma ( h ), or prekallikrein-deficient human plasma with exogenous human PKal added ( i ). j , Quantification of adipose conversion; absorbance is designated as 1.0 for cells grown in NHP. Data are means ± s.d.

    Techniques Used: Incubation, Mass Spectrometry, Western Blot, Staining

    PKal-mediated Plg activation promotes fibronectin degradation during adipocyte differentiation a , PKal activates Plg at physiologically relevant concentrations. Western blot showing generation of the M r 25K protease domain (PD) of plasmin at 5, 10 and 15 min. b , Activation of Plg in the conditioned medium of 3T3-L1 cells is enhanced in the presence of PKal. Western blot of conditioned media from 3T3-L1 cells differentiated in the absence of FBS but with exogenous plasminogen (Plg), plasma kallikrein (PKal) or PAI-1, showing generation of plasmin (Pls) and the protease domain. c , Western blot (against human fibronectin) showing fibronectin (FN) cleavage products after incubation with serine proteases. d , Western blot (against rat fibronectin) of whole-cell lysates from preadipocytes (Pre), differentiated adipocytes (Adi) and cells differentiated in the presence of WT ecotin (EcoWT), showing that fibronectin is present in lysates from 3T3-L1 preadipocytes but is downregulated in adipocytes. e , Western blot (against endogenous mouse fibronectin) of conditioned media from preadipocytes, differentiated adipocytes, and cells differentiated in the presence of WT ecotin (EcoWT) and EcoRR, showing that fibronectin is cleaved during adipocyte differentiation. f , Western blot showing generation of the protease domain of plasmin at 5, 10 and 15 min in the absence (Control) and presence of fibrin and fibronectin. g–l , Staining with Oil red O of preadipocytes ( g ), of adipocytes ( h ) and of 3T3-L1 cells differentiated in the presence of fibronectin ( i ), fibronectin matrix and cytochalasin D ( j ), WT ecotin ( k ), or WT ecotin and cytochalasin D ( l ).
    Figure Legend Snippet: PKal-mediated Plg activation promotes fibronectin degradation during adipocyte differentiation a , PKal activates Plg at physiologically relevant concentrations. Western blot showing generation of the M r 25K protease domain (PD) of plasmin at 5, 10 and 15 min. b , Activation of Plg in the conditioned medium of 3T3-L1 cells is enhanced in the presence of PKal. Western blot of conditioned media from 3T3-L1 cells differentiated in the absence of FBS but with exogenous plasminogen (Plg), plasma kallikrein (PKal) or PAI-1, showing generation of plasmin (Pls) and the protease domain. c , Western blot (against human fibronectin) showing fibronectin (FN) cleavage products after incubation with serine proteases. d , Western blot (against rat fibronectin) of whole-cell lysates from preadipocytes (Pre), differentiated adipocytes (Adi) and cells differentiated in the presence of WT ecotin (EcoWT), showing that fibronectin is present in lysates from 3T3-L1 preadipocytes but is downregulated in adipocytes. e , Western blot (against endogenous mouse fibronectin) of conditioned media from preadipocytes, differentiated adipocytes, and cells differentiated in the presence of WT ecotin (EcoWT) and EcoRR, showing that fibronectin is cleaved during adipocyte differentiation. f , Western blot showing generation of the protease domain of plasmin at 5, 10 and 15 min in the absence (Control) and presence of fibrin and fibronectin. g–l , Staining with Oil red O of preadipocytes ( g ), of adipocytes ( h ) and of 3T3-L1 cells differentiated in the presence of fibronectin ( i ), fibronectin matrix and cytochalasin D ( j ), WT ecotin ( k ), or WT ecotin and cytochalasin D ( l ).

    Techniques Used: Activation Assay, Western Blot, Incubation, Staining

    Inhibition of serine proteases during differentiation of 3T3-L1 cells reduces adipose conversion a , e–h , Staining of cells with Oil red O to visualize the extent of adipose conversion. b , i , Quantification of lipid accumulation; absorbance is designated as 1.0 for adipocytes. Data are means ± s.d. c , d , Western blotting of 3T3-L1 nuclear lysates to detect C/EBPβ ( c ) and PPARγ ( d ). Pre, 3T3-L1 preadipocytes; Adi, adipocytes, EcoWT, differentiating cells treated with WT ecotin; EcoRR, differentiating cells treated with EcoRR. Where indicated, adipocytes were treated with PAI-1 or α-2-antiplasmin (α2-AP).
    Figure Legend Snippet: Inhibition of serine proteases during differentiation of 3T3-L1 cells reduces adipose conversion a , e–h , Staining of cells with Oil red O to visualize the extent of adipose conversion. b , i , Quantification of lipid accumulation; absorbance is designated as 1.0 for adipocytes. Data are means ± s.d. c , d , Western blotting of 3T3-L1 nuclear lysates to detect C/EBPβ ( c ) and PPARγ ( d ). Pre, 3T3-L1 preadipocytes; Adi, adipocytes, EcoWT, differentiating cells treated with WT ecotin; EcoRR, differentiating cells treated with EcoRR. Where indicated, adipocytes were treated with PAI-1 or α-2-antiplasmin (α2-AP).

    Techniques Used: Inhibition, Staining, Western Blot

    18) Product Images from "Adipogenesis is differentially impaired by thyroid hormone receptor mutant isoforms"

    Article Title: Adipogenesis is differentially impaired by thyroid hormone receptor mutant isoforms

    Journal: Journal of molecular endocrinology

    doi: 10.1677/JME-09-0137

    )). Lane 3 indicates control cells that were used as negative controls, indicating the specific bands detected in lanes 1 and 2. GAPDH was used as a loading control. (B) Total cellular lysates (30 μg) were used in the western blot analysis. Endogenous TRα1 and TRβ1 receptor proteins in L1-α1PV cells (lane 1), L1-β1PV cells (lane 2), and 3T3-L1 cells (lane 3) were detected by monoclonal antibody C4 that recognizes the C-terminus ofTRβ1 and TRα1 receptors. GAPDH wasusedasa loading control.
    Figure Legend Snippet: )). Lane 3 indicates control cells that were used as negative controls, indicating the specific bands detected in lanes 1 and 2. GAPDH was used as a loading control. (B) Total cellular lysates (30 μg) were used in the western blot analysis. Endogenous TRα1 and TRβ1 receptor proteins in L1-α1PV cells (lane 1), L1-β1PV cells (lane 2), and 3T3-L1 cells (lane 3) were detected by monoclonal antibody C4 that recognizes the C-terminus ofTRβ1 and TRα1 receptors. GAPDH wasusedasa loading control.

    Techniques Used: Western Blot

    T 3 -induced adipogenesis in 3T3-L1 cells is differentially impaired by TR mutant isoforms. Control 3T3-L1, L1-β1PV, and L1-α1PV cells were induced to undergo adipogenesis in the absence (panels 1, 2, and 3) or presence of T 3 (2 nM, panels 4, 5, and 6) as described in Materials and methods. After 7 days, matured adipocytes with lipid droplets were visualized by staining with Oil Red O (A) and by phase contrast microscopy (C). (B) Quantification of relative Oil Red O staining intensities of lipid droplets in matured adipocytes. Data are expressed as mean±S.E.M. ( n =6). The P values are indicated.
    Figure Legend Snippet: T 3 -induced adipogenesis in 3T3-L1 cells is differentially impaired by TR mutant isoforms. Control 3T3-L1, L1-β1PV, and L1-α1PV cells were induced to undergo adipogenesis in the absence (panels 1, 2, and 3) or presence of T 3 (2 nM, panels 4, 5, and 6) as described in Materials and methods. After 7 days, matured adipocytes with lipid droplets were visualized by staining with Oil Red O (A) and by phase contrast microscopy (C). (B) Quantification of relative Oil Red O staining intensities of lipid droplets in matured adipocytes. Data are expressed as mean±S.E.M. ( n =6). The P values are indicated.

    Techniques Used: Mutagenesis, Staining, Microscopy

    19) Product Images from "CCAAT/enhancer-binding protein-β functions as a negative regulator of Wnt/β-catenin signaling through activation of AXIN1 gene expression"

    Article Title: CCAAT/enhancer-binding protein-β functions as a negative regulator of Wnt/β-catenin signaling through activation of AXIN1 gene expression

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1072-1

    Activation of AXIN1 gene expression during adipogenesis. a 3T3-L1 cell lysates were prepared at 8, 16, and 24 h after MDI treatment and subjected to western blot analysis using the indicated antibodies. b Real-time PCR for the analysis of β-catenin and β-actin was performed with total RNA prepared from 3T3-L1 cells treated with MDI for 24 h. c Cell lysates prepared from 3T3-L1 cells treated with MDI for 24 h were subjected to western blot analysis against the indicated antibodies. Identical amount of β-catenin was loaded in each lane. d Cytosolic proteins prepared from MDI-treated 3T3-L1 cells exposed to MG132 (10 μM) were subjected to western blot analysis against the indicated antibodies. e Real-time PCR for Axin1 and C/EBP-β expression was performed with total RNA prepared from 3T3-L1 cells treated with MDI for 24 h. In b , e , results are the average of three experiments, and bars indicate standard deviations. * P
    Figure Legend Snippet: Activation of AXIN1 gene expression during adipogenesis. a 3T3-L1 cell lysates were prepared at 8, 16, and 24 h after MDI treatment and subjected to western blot analysis using the indicated antibodies. b Real-time PCR for the analysis of β-catenin and β-actin was performed with total RNA prepared from 3T3-L1 cells treated with MDI for 24 h. c Cell lysates prepared from 3T3-L1 cells treated with MDI for 24 h were subjected to western blot analysis against the indicated antibodies. Identical amount of β-catenin was loaded in each lane. d Cytosolic proteins prepared from MDI-treated 3T3-L1 cells exposed to MG132 (10 μM) were subjected to western blot analysis against the indicated antibodies. e Real-time PCR for Axin1 and C/EBP-β expression was performed with total RNA prepared from 3T3-L1 cells treated with MDI for 24 h. In b , e , results are the average of three experiments, and bars indicate standard deviations. * P

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

    Identification of C/EBP-β as a bona fide transcriptional activator of AXIN1 gene expression. a , b HEK293 cells were transfected with indicated Axin1 promoter constructs, C/EBP-β expression plasmid and pRL-CMV plasmid for 48 h. Luciferase activities were measured 48 h after transfection using the Dual Luciferase Reporter Assay System (Promega). TOPFlash activity is reported as relative light units (RLUs) normalized to Renilla luciferase activity. c 3T3-L1 cells were transfected with mAxin-1013 promoter constructs and C/EBP-β expression plasmid and pRL-CMV plasmid for 48 h. d – f HEK293 cells were transfected with pcDNA or C/EBP-β expression plasmids for 48 h. Total RNA was subjected to real-time PCR for Axin1 expression ( d ), and whole-cell lysates were subjected to western blot analysis and probed with the indicated antibodies ( e ). Chromatin samples were prepared from HEK293 cells and subjected to ChIP analysis with anti-C/EBP-β antibody or control IgG antibody. The amounts of immunoprecipitated C/EBP-β promoter regions were quantified by real-time PCR ( f ). g Chromatin samples were prepared from 3T3-L1 cells treated with MDI for 24 h and subjected to ChIP analysis with anti-C/EBP-β antibody or control IgG antibody. In a–d , f and g , results are the average of three experiments, and bars indicate standard deviations. * P
    Figure Legend Snippet: Identification of C/EBP-β as a bona fide transcriptional activator of AXIN1 gene expression. a , b HEK293 cells were transfected with indicated Axin1 promoter constructs, C/EBP-β expression plasmid and pRL-CMV plasmid for 48 h. Luciferase activities were measured 48 h after transfection using the Dual Luciferase Reporter Assay System (Promega). TOPFlash activity is reported as relative light units (RLUs) normalized to Renilla luciferase activity. c 3T3-L1 cells were transfected with mAxin-1013 promoter constructs and C/EBP-β expression plasmid and pRL-CMV plasmid for 48 h. d – f HEK293 cells were transfected with pcDNA or C/EBP-β expression plasmids for 48 h. Total RNA was subjected to real-time PCR for Axin1 expression ( d ), and whole-cell lysates were subjected to western blot analysis and probed with the indicated antibodies ( e ). Chromatin samples were prepared from HEK293 cells and subjected to ChIP analysis with anti-C/EBP-β antibody or control IgG antibody. The amounts of immunoprecipitated C/EBP-β promoter regions were quantified by real-time PCR ( f ). g Chromatin samples were prepared from 3T3-L1 cells treated with MDI for 24 h and subjected to ChIP analysis with anti-C/EBP-β antibody or control IgG antibody. In a–d , f and g , results are the average of three experiments, and bars indicate standard deviations. * P

    Techniques Used: Expressing, Transfection, Construct, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation

    20) Product Images from "Oncogenic steroid receptor coactivator-3 is a key regulator of the white adipogenic program"

    Article Title: Oncogenic steroid receptor coactivator-3 is a key regulator of the white adipogenic program

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

    doi: 10.1073/pnas.0608711103

    Increased nuclear levels of SRC-3 in the early steps of adipocyte differentiation and accumulation to high levels in the nucleus of differentiated fat cells. ( A ) Immunofluorescence staining of SRC-3 protein in intact 3T3-L1 cells in the early steps of adipocyte differentiation. The fluorescent dye Bodipy and DAPI were, respectively, used to stain the lipids contained in the droplets of differentiated fat cells and nuclei of existing cells. ( B ) Quantitative analysis of nuclear fluorescence intensity in the early stages of 3T3-L1 fat-cell differentiation. ( C ) Western blot analysis of SRC-3 protein levels in 3T3-L1 differentiating cells in the early steps of adipocyte differentiation. α-tubulin served as a loading control. ( D ) Immunofluorescence staining of SRC-3 protein in intact 3T3-L1 cells in the late steps of adipocyte differentiation (day 4). [Magnification: ×100 ( Upper ); ×40 ( Lower ).] Arrows indicate that cells showing high SRC-3 protein levels in the nucleus have intense cytoplasmic staining of lipid droplets. ( E ) Quantification of differentiated 3T3-L1 fat cells showing strong nuclear localization of SRC-3. One hundred differentiated cells showing intense lipid droplet staining (size > 2 μm) were counted manually for either strong (nuclear fluorescence value > 2 × 10 3 ) or weak nuclear SRC-3 fluorescence intensity (nuclear fluorescence value lower than 2 × 10 3 ). Errors bars represent S.E.M.
    Figure Legend Snippet: Increased nuclear levels of SRC-3 in the early steps of adipocyte differentiation and accumulation to high levels in the nucleus of differentiated fat cells. ( A ) Immunofluorescence staining of SRC-3 protein in intact 3T3-L1 cells in the early steps of adipocyte differentiation. The fluorescent dye Bodipy and DAPI were, respectively, used to stain the lipids contained in the droplets of differentiated fat cells and nuclei of existing cells. ( B ) Quantitative analysis of nuclear fluorescence intensity in the early stages of 3T3-L1 fat-cell differentiation. ( C ) Western blot analysis of SRC-3 protein levels in 3T3-L1 differentiating cells in the early steps of adipocyte differentiation. α-tubulin served as a loading control. ( D ) Immunofluorescence staining of SRC-3 protein in intact 3T3-L1 cells in the late steps of adipocyte differentiation (day 4). [Magnification: ×100 ( Upper ); ×40 ( Lower ).] Arrows indicate that cells showing high SRC-3 protein levels in the nucleus have intense cytoplasmic staining of lipid droplets. ( E ) Quantification of differentiated 3T3-L1 fat cells showing strong nuclear localization of SRC-3. One hundred differentiated cells showing intense lipid droplet staining (size > 2 μm) were counted manually for either strong (nuclear fluorescence value > 2 × 10 3 ) or weak nuclear SRC-3 fluorescence intensity (nuclear fluorescence value lower than 2 × 10 3 ). Errors bars represent S.E.M.

    Techniques Used: Immunofluorescence, Staining, Fluorescence, Cell Differentiation, Western Blot

    21) Product Images from "Mitogen-Activated Protein Kinase Phosphatase 3 (MKP-3)–Deficient Mice Are Resistant to Diet-Induced Obesity"

    Article Title: Mitogen-Activated Protein Kinase Phosphatase 3 (MKP-3)–Deficient Mice Are Resistant to Diet-Induced Obesity

    Journal: Diabetes

    doi: 10.2337/db14-0066

    MKP-3 is involved in adipogenesis. A : MKP-3 mRNA expression in WAT in male mice fed an HFD or chow ( n = 4 per group). B : MKP-3 protein expression in WAT of mice fed an HFD or chow ( n = 4 per group). C : MKP-3 protein expression in 3T3-L1 cells 2 days after induction (D2PI) vs. preadipocytes (pre). D and E : MKP-3 knockdown in 3T3-L1 CAR preadipocytes and the effect on adipogenesis. F : Gene expression pattern during adipogenesis of stromal vascular cells isolated from WT or MKP-3 −/− mice ( n = 3–4 per group). For cell-based experiments, results shown here are representative of 3 independent experiments. * P
    Figure Legend Snippet: MKP-3 is involved in adipogenesis. A : MKP-3 mRNA expression in WAT in male mice fed an HFD or chow ( n = 4 per group). B : MKP-3 protein expression in WAT of mice fed an HFD or chow ( n = 4 per group). C : MKP-3 protein expression in 3T3-L1 cells 2 days after induction (D2PI) vs. preadipocytes (pre). D and E : MKP-3 knockdown in 3T3-L1 CAR preadipocytes and the effect on adipogenesis. F : Gene expression pattern during adipogenesis of stromal vascular cells isolated from WT or MKP-3 −/− mice ( n = 3–4 per group). For cell-based experiments, results shown here are representative of 3 independent experiments. * P

    Techniques Used: Expressing, Mouse Assay, Isolation

    22) Product Images from "Anandamide-derived Prostamide F2α"

    Article Title: Anandamide-derived Prostamide F2α

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.489906

    Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α
    Figure Legend Snippet: Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ
    Figure Legend Snippet: Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ

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

    Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ
    Figure Legend Snippet: Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ

    Techniques Used: Inhibition, Activity Assay, Real-time Polymerase Chain Reaction

    Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence
    Figure Legend Snippet: Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence

    Techniques Used: Mass Spectrometry

    23) Product Images from "CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP) Regulates Osteoblast Differentiation"

    Article Title: CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP) Regulates Osteoblast Differentiation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.02429-05

    CHOP suppresses adipocyte differentiation. (A) Calvarial osteoblasts were infected with LacZ (MOI = 200), FLAG-CHOP (F-CHOP) (MOI = 200), or FLAG-C/EBPβ (MOI = 100) adenovirus and cultured for 3 days. Then, nuclear extracts from infected cells were immunoblotted with anti-FLAG antibody to determine the expression levels of induced proteins. (B) 3T3-L1 cells were infected with LacZ adenovirus, C/EBPβ adenovirus, or both at an MOI of 100 for each and incubated with or without DIII cocktail for 4 days. Then, cells were examined for adipogenic differentiation by Nile red staining and real-time RT-PCR for aP2 and adipsin. mRNA values were normalized to the amounts of HPRT1. (C) 3T3-L1 cells were infected with LacZ adenovirus, FLAG-C/EBPβ adenovirus, or both at an MOI of 100 for each and incubated for 14 days. Then, expression levels of PPARγ2 and adipsin were assessed by real-time RT-PCR. mRNA values were normalized to the amounts of HPRT1.
    Figure Legend Snippet: CHOP suppresses adipocyte differentiation. (A) Calvarial osteoblasts were infected with LacZ (MOI = 200), FLAG-CHOP (F-CHOP) (MOI = 200), or FLAG-C/EBPβ (MOI = 100) adenovirus and cultured for 3 days. Then, nuclear extracts from infected cells were immunoblotted with anti-FLAG antibody to determine the expression levels of induced proteins. (B) 3T3-L1 cells were infected with LacZ adenovirus, C/EBPβ adenovirus, or both at an MOI of 100 for each and incubated with or without DIII cocktail for 4 days. Then, cells were examined for adipogenic differentiation by Nile red staining and real-time RT-PCR for aP2 and adipsin. mRNA values were normalized to the amounts of HPRT1. (C) 3T3-L1 cells were infected with LacZ adenovirus, FLAG-C/EBPβ adenovirus, or both at an MOI of 100 for each and incubated for 14 days. Then, expression levels of PPARγ2 and adipsin were assessed by real-time RT-PCR. mRNA values were normalized to the amounts of HPRT1.

    Techniques Used: Infection, Cell Culture, Expressing, Incubation, Staining, Quantitative RT-PCR

    Profiles of expression of C/EBP family members in neonatal tissues and mesenchymal progenitor cells. (A) RT-PCR performed on cDNA derived from various neonatal mouse tissues. RT-PCR experiments were performed using primers specific for CHOP, C/EBPα, C/EBPβ, C/EBPδ, and HPRT1. Amplification of CHOP, C/EBPα, C/EBPβ, or C/EBPδ was performed with 32 cycles. Amplification of a HPRT1 fragment was used to confirm equal levels of target cDNA in the samples. (B) RT-PCR performed on cDNA derived from several mesenchymal cells cultured with or without BMP-2 (300 ng/ml) for 5 days or from 3T3-L1 cells cultured with or without adipogenic cocktail DIII for 2 days. Amplification of CHOP, C/EBPα, C/EBPβ, or C/EBPδ was performed with 30 cycles. MSC, marrow stromal cells. (C) C2C12 cells were cultured with or without BMP-2 (300 ng/ml) for 4 days, and total cell lysates were immunoblotted with anti-GADD153 (anti-CHOP) antibody, anti-C/EBPβ antibody, or antitubulin antibody. Lysate of 3T3-L1 cells which express CHOP and C/EBPβ was loaded as a positive control. The asterisk indicates a nonspecific band.
    Figure Legend Snippet: Profiles of expression of C/EBP family members in neonatal tissues and mesenchymal progenitor cells. (A) RT-PCR performed on cDNA derived from various neonatal mouse tissues. RT-PCR experiments were performed using primers specific for CHOP, C/EBPα, C/EBPβ, C/EBPδ, and HPRT1. Amplification of CHOP, C/EBPα, C/EBPβ, or C/EBPδ was performed with 32 cycles. Amplification of a HPRT1 fragment was used to confirm equal levels of target cDNA in the samples. (B) RT-PCR performed on cDNA derived from several mesenchymal cells cultured with or without BMP-2 (300 ng/ml) for 5 days or from 3T3-L1 cells cultured with or without adipogenic cocktail DIII for 2 days. Amplification of CHOP, C/EBPα, C/EBPβ, or C/EBPδ was performed with 30 cycles. MSC, marrow stromal cells. (C) C2C12 cells were cultured with or without BMP-2 (300 ng/ml) for 4 days, and total cell lysates were immunoblotted with anti-GADD153 (anti-CHOP) antibody, anti-C/EBPβ antibody, or antitubulin antibody. Lysate of 3T3-L1 cells which express CHOP and C/EBPβ was loaded as a positive control. The asterisk indicates a nonspecific band.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Amplification, Cell Culture, Positive Control

    24) Product Images from "Zinc-?2-glycoprotein, a lipid mobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia"

    Article Title: Zinc-?2-glycoprotein, a lipid mobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia

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

    doi: 10.1073/pnas.0308647100

    ZAG gene expression in 3T3-L1 adipocytes by real-time RT-PCR. ( A ) Expression during differentiation of 3T3-L1 cells into adipocytes. Data are means ± SEM, relative to day 0 ( n = 3). ( B ) Effects of BRL 37344 or dexamethasone (DEX) on ZAG mRNA levels in 3T3-L1 adipocytes. Cells were taken at day 8 after induction of differentiation and incubated for 24 h with BRL 37344 (1μM) or dexamethasone (20 nM). ZAG mRNA levels were normalized to β-actin. Data are means ± SEM ( n = 6); changes are relative to controls. * , P
    Figure Legend Snippet: ZAG gene expression in 3T3-L1 adipocytes by real-time RT-PCR. ( A ) Expression during differentiation of 3T3-L1 cells into adipocytes. Data are means ± SEM, relative to day 0 ( n = 3). ( B ) Effects of BRL 37344 or dexamethasone (DEX) on ZAG mRNA levels in 3T3-L1 adipocytes. Cells were taken at day 8 after induction of differentiation and incubated for 24 h with BRL 37344 (1μM) or dexamethasone (20 nM). ZAG mRNA levels were normalized to β-actin. Data are means ± SEM ( n = 6); changes are relative to controls. * , P

    Techniques Used: Expressing, Quantitative RT-PCR, Incubation

    ZAG gene expression in mouse tissues and adipocytes examined by RT-PCR. ( A ) Tissue specificity of expression. ( B ) Expression in fat depots. ( C ) Expression in mature adipocytes and stromal-vascular cells (isolated by collagenase digestion) and 3T3-L1 adipocytes. The 3T3-L1 cells were taken at days 3 and 6 after inducing differentiation. HPRT, hypoxanthine-phosphoribosyl transferase; e-WAT, epididymal WAT; peri, perirenal; sub, s.c.; sk, skeletal; S-V, stromal vascular.
    Figure Legend Snippet: ZAG gene expression in mouse tissues and adipocytes examined by RT-PCR. ( A ) Tissue specificity of expression. ( B ) Expression in fat depots. ( C ) Expression in mature adipocytes and stromal-vascular cells (isolated by collagenase digestion) and 3T3-L1 adipocytes. The 3T3-L1 cells were taken at days 3 and 6 after inducing differentiation. HPRT, hypoxanthine-phosphoribosyl transferase; e-WAT, epididymal WAT; peri, perirenal; sub, s.c.; sk, skeletal; S-V, stromal vascular.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

    25) Product Images from "Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling"

    Article Title: Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling

    Journal: Molecular and cellular endocrinology

    doi: 10.1016/j.mce.2015.06.036

    DEX induces the change in NCoR mRNA splicing observed during 3T3-L1 cells differentiation. Cells were differentiated with various combinations of the individual factors within the tripartite differentiation cocktail. Means ± S.E.M. (n = 3 for SMRT Exon 40 and NCoR Exon 28, and n = 5 for NCoR Exon 37) are presented. p
    Figure Legend Snippet: DEX induces the change in NCoR mRNA splicing observed during 3T3-L1 cells differentiation. Cells were differentiated with various combinations of the individual factors within the tripartite differentiation cocktail. Means ± S.E.M. (n = 3 for SMRT Exon 40 and NCoR Exon 28, and n = 5 for NCoR Exon 37) are presented. p

    Techniques Used:

    26) Product Images from "The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria *"

    Article Title: The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.727248

    Deletion of ATP7A in 3T3-L1 preadipocytes is associated with elevation of cellular copper content and increased oxidation of glutathione in mitochondria. A , CRISPR/Cas9 targeting of exon 2 generated deletion in both alleles of genomic ATP7A in 3T3-L1 cells. B , Western blotting analysis of cell lysates shows the presence of ATP7A in the WT 3T3-L1 cells and a loss of ATP7A expression in CRISPR/Cas9-targeted 3T3-L1 cells. α-Tubulin was used as a loading control. C , copper levels in wild-type (3.4 ± 0.49 ng/mg protein) and ATP7A −/− 3T3-L1 (10.32 ± 0.41 ng/mg protein) cells under basal conditions (****, p
    Figure Legend Snippet: Deletion of ATP7A in 3T3-L1 preadipocytes is associated with elevation of cellular copper content and increased oxidation of glutathione in mitochondria. A , CRISPR/Cas9 targeting of exon 2 generated deletion in both alleles of genomic ATP7A in 3T3-L1 cells. B , Western blotting analysis of cell lysates shows the presence of ATP7A in the WT 3T3-L1 cells and a loss of ATP7A expression in CRISPR/Cas9-targeted 3T3-L1 cells. α-Tubulin was used as a loading control. C , copper levels in wild-type (3.4 ± 0.49 ng/mg protein) and ATP7A −/− 3T3-L1 (10.32 ± 0.41 ng/mg protein) cells under basal conditions (****, p

    Techniques Used: CRISPR, Generated, Western Blot, Expressing

    27) Product Images from "ER stress increases StarD5 expression by stabilizing its mRNA and leads to relocalization of its protein from the nucleus to the membranes"

    Article Title: ER stress increases StarD5 expression by stabilizing its mRNA and leads to relocalization of its protein from the nucleus to the membranes

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M031997

    Increased StarD5 mRNA is maintained throughout ER stress induction. ER stress was induced in 3T3-L1 cells by the addition of 2 μM Tg as detailed in Materials and Methods. Total RNA was isolated, and BiP, ATF4, CHOP, and StarD5 mRNAs were quantified
    Figure Legend Snippet: Increased StarD5 mRNA is maintained throughout ER stress induction. ER stress was induced in 3T3-L1 cells by the addition of 2 μM Tg as detailed in Materials and Methods. Total RNA was isolated, and BiP, ATF4, CHOP, and StarD5 mRNAs were quantified

    Techniques Used: Isolation

    Tg stabilizes StarD5 mRNA in 3T3-L1 cells. 3T3-L1 cells were treated with 2 μM Tg, as indicated in Materials and Methods, in the presence of actinomycin D (5 μg/ml). As controls, cells were left untreated or treated with DMSO as indicated.
    Figure Legend Snippet: Tg stabilizes StarD5 mRNA in 3T3-L1 cells. 3T3-L1 cells were treated with 2 μM Tg, as indicated in Materials and Methods, in the presence of actinomycin D (5 μg/ml). As controls, cells were left untreated or treated with DMSO as indicated.

    Techniques Used:

    StarD5 protein redistributes to the cytosol and to membranes following induction of ER stress in 3T3-L1 cells. 3T3-L1 cells were cultured on cover slips in the absence or presence of 2 μM Tg for 6 h as indicated. A: The cells were analyzed by
    Figure Legend Snippet: StarD5 protein redistributes to the cytosol and to membranes following induction of ER stress in 3T3-L1 cells. 3T3-L1 cells were cultured on cover slips in the absence or presence of 2 μM Tg for 6 h as indicated. A: The cells were analyzed by

    Techniques Used: Cell Culture

    ER stress causes intracellular cholesterol accumulation due to an increase in HMG-CoA reductase expression in 3T3-L1 cells. 3T3-L1 cells were treated with Tg in the absence and presence of mevinolin for the times shown as indicated in Materials and Methods.
    Figure Legend Snippet: ER stress causes intracellular cholesterol accumulation due to an increase in HMG-CoA reductase expression in 3T3-L1 cells. 3T3-L1 cells were treated with Tg in the absence and presence of mevinolin for the times shown as indicated in Materials and Methods.

    Techniques Used: Expressing

    StarD5 expression is increased by ER stress. A: ER stress was induced in 3T3-L1 cells by the addition of 2 μM Tg or 5 μg/ml Tu as indicated and detailed in Materials and Methods. Twenty four hours after the additions, total RNA was isolated,
    Figure Legend Snippet: StarD5 expression is increased by ER stress. A: ER stress was induced in 3T3-L1 cells by the addition of 2 μM Tg or 5 μg/ml Tu as indicated and detailed in Materials and Methods. Twenty four hours after the additions, total RNA was isolated,

    Techniques Used: Expressing, Isolation

    28) 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 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

    29) Product Images from "LSD1 promotes oxidative metabolism of white adipose tissue"

    Article Title: LSD1 promotes oxidative metabolism of white adipose tissue

    Journal: Nature communications

    doi: 10.1038/ncomms5093

    LSD1 demethylase activity is essential for early adipogenesis but dispensable at later stages of differentiation a, Time scale of treatment of primary adipocytes, C3H-10T1/2, or 3T3-L1 cells with LSD1-specific inhibiter GSK690. b, Western blot analysis of LSD1 and Fabp4 of differentiated primary adipocytes treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. β-Tubulin served as a loading control. c, Relative transcript levels of indicated markers in differentiated primary adipocytes treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. d, Oil red O staining of 3T3-L1, C3H-10T1/2, or differentiated primary adipocytes treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. Magnification: 200 x, scale bar represents 100 μm. e, Western blot analysis of LSD1 and Fabp4 of (left panel) C3H-10T1/2 or (right panel) 3T3-L1 cells treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. β-Tubulin served as a loading control. f, Relative transcript levels of the indicated markers in C3H-10T1/2 treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. b-f : n = 9. Standard deviation represents + s.e.m. Experiments ( c,f ) were independently repeated at least three times in triplicate. Statistical analysis was performed using two-tailed Student’s t-test. * p
    Figure Legend Snippet: LSD1 demethylase activity is essential for early adipogenesis but dispensable at later stages of differentiation a, Time scale of treatment of primary adipocytes, C3H-10T1/2, or 3T3-L1 cells with LSD1-specific inhibiter GSK690. b, Western blot analysis of LSD1 and Fabp4 of differentiated primary adipocytes treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. β-Tubulin served as a loading control. c, Relative transcript levels of indicated markers in differentiated primary adipocytes treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. d, Oil red O staining of 3T3-L1, C3H-10T1/2, or differentiated primary adipocytes treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. Magnification: 200 x, scale bar represents 100 μm. e, Western blot analysis of LSD1 and Fabp4 of (left panel) C3H-10T1/2 or (right panel) 3T3-L1 cells treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. β-Tubulin served as a loading control. f, Relative transcript levels of the indicated markers in C3H-10T1/2 treated with LSD1-specific inhibiter GSK690 or vehicle at the indicated time points of differentiation. b-f : n = 9. Standard deviation represents + s.e.m. Experiments ( c,f ) were independently repeated at least three times in triplicate. Statistical analysis was performed using two-tailed Student’s t-test. * p

    Techniques Used: Activity Assay, Western Blot, Staining, Standard Deviation, Two Tailed Test

    LSD1 expression is induced in white fat pads after cold exposure or β3-adrenergic treatment of mice a,b, Western blot analysis of LSD1 and Ucp1 in inguinal (ing) white adipose tissue (WAT) of mice ( a ) maintained at either 24 or 10 °C, or ( b ) treated with vehicle or the β3-adrenergic agonist CL316,243. β-Tubulin was used as a loading control. c,d, Relative transcript levels of the indicated markers in differentiated (day 7) ( c ) C3H-10T1/2 or ( d ) 3T3-L1 cells treated with vehicle or CL316,243 and transfected with unrelated control siRNA (siCtrl) or siRNA directed against LSD1 (siLSD1). a,b: n = 10, c,d: n = 9. Standard deviation represents + s.e.m. Experiments ( c,d ) were independently repeated at least three times in triplicate. Statistical analysis was performed using two-tailed Student’s t-test. * p
    Figure Legend Snippet: LSD1 expression is induced in white fat pads after cold exposure or β3-adrenergic treatment of mice a,b, Western blot analysis of LSD1 and Ucp1 in inguinal (ing) white adipose tissue (WAT) of mice ( a ) maintained at either 24 or 10 °C, or ( b ) treated with vehicle or the β3-adrenergic agonist CL316,243. β-Tubulin was used as a loading control. c,d, Relative transcript levels of the indicated markers in differentiated (day 7) ( c ) C3H-10T1/2 or ( d ) 3T3-L1 cells treated with vehicle or CL316,243 and transfected with unrelated control siRNA (siCtrl) or siRNA directed against LSD1 (siLSD1). a,b: n = 10, c,d: n = 9. Standard deviation represents + s.e.m. Experiments ( c,d ) were independently repeated at least three times in triplicate. Statistical analysis was performed using two-tailed Student’s t-test. * p

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

    30) Product Images from "Heparan sulfate promotes differentiation of white adipocytes to maintain insulin sensitivity and glucose homeostasis"

    Article Title: Heparan sulfate promotes differentiation of white adipocytes to maintain insulin sensitivity and glucose homeostasis

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.101006

    Insulin-dependent glucose uptake was decreased in Ext1 -heterozygous knockout 3T3-L1 ( Ext1 +/− ) cells. A , left , representative images of Western blot for phosphorylated insulin receptor substrate (pIrs), Irs, pAkt, and Akt, with or without insulin stimulation. Middle , protein levels of pIrs corrected by Irs intensity, with or without insulin stimulation. n = 3. Right , the protein levels of pAkt corrected by Akt intensity, with or without insulin stimulation. n = 3. The mean intensity of control 3T3-L1 cells with insulin was set to 1. B , measurement of deoxy- d -glucose uptake in 3T3-L1 cells, with or without insulin stimulation. Gray : control cells, white : Ext1 +/− cells. n = 12. ∗ p
    Figure Legend Snippet: Insulin-dependent glucose uptake was decreased in Ext1 -heterozygous knockout 3T3-L1 ( Ext1 +/− ) cells. A , left , representative images of Western blot for phosphorylated insulin receptor substrate (pIrs), Irs, pAkt, and Akt, with or without insulin stimulation. Middle , protein levels of pIrs corrected by Irs intensity, with or without insulin stimulation. n = 3. Right , the protein levels of pAkt corrected by Akt intensity, with or without insulin stimulation. n = 3. The mean intensity of control 3T3-L1 cells with insulin was set to 1. B , measurement of deoxy- d -glucose uptake in 3T3-L1 cells, with or without insulin stimulation. Gray : control cells, white : Ext1 +/− cells. n = 12. ∗ p

    Techniques Used: Knock-Out, Western Blot

    Heparan sulfate (HS) plays important roles in 3T3-L1 differentiation via modulating BMP4–FGF1 signaling. A , observation of 3T3-L1 cells at 0 and 7 days after inducing differentiation. The white scale bar represents 100 μm. B , relative mRNA expression levels of differentiation markers at 0 and 7 days after inducing differentiation. n = 3–8. The mean mRNA expression level of control 3T3-L1 cells at day 0 was set to 1. Gray : control cells at day 0; red : control cells at day 7; white : Ext1 +/− cells at day 0; blue : Ext1 +/− cells at day 7. ∗Comparison of gray and red ; † comparison of white and blue ; # comparison of red and blue . C , relative mRNA expression levels of differentiation-related genes in control 3T3-L1 cells. n = 4–6. Gray and white bars indicate the day after inducing differentiation. The mean mRNA expression level at day 0 was set to 1. D , observation of 3T3-L1 cells at 7 days after inducing differentiation, with or without BMP4–FGF1 treatment. Upper pictures : without BMP4–FGF1 treatment; lower pictures : with BMP4–FGF1 treatment. The white scale bar represents 100 μm. E , relative mRNA expression levels of BMP4–FGF1 signaling and differentiation-related genes. n = 6–9. The mean mRNA expression level of control 3T3-L1 cells without BMP4–FGF1 treatment was set to 1. Gray : control cells without BMP4–FGF1 treatment; red : control cells with BMP4–FGF1 treatment; white : Ext1 +/− cells without BMP4–FGF1 treatment; blue : Ext1 +/− cells with BMP4–FGF1 treatment. ∗Comparison of gray and red ; † Comparison of gray and white ; # Comparison of white and blue . ∗ ,# p
    Figure Legend Snippet: Heparan sulfate (HS) plays important roles in 3T3-L1 differentiation via modulating BMP4–FGF1 signaling. A , observation of 3T3-L1 cells at 0 and 7 days after inducing differentiation. The white scale bar represents 100 μm. B , relative mRNA expression levels of differentiation markers at 0 and 7 days after inducing differentiation. n = 3–8. The mean mRNA expression level of control 3T3-L1 cells at day 0 was set to 1. Gray : control cells at day 0; red : control cells at day 7; white : Ext1 +/− cells at day 0; blue : Ext1 +/− cells at day 7. ∗Comparison of gray and red ; † comparison of white and blue ; # comparison of red and blue . C , relative mRNA expression levels of differentiation-related genes in control 3T3-L1 cells. n = 4–6. Gray and white bars indicate the day after inducing differentiation. The mean mRNA expression level at day 0 was set to 1. D , observation of 3T3-L1 cells at 7 days after inducing differentiation, with or without BMP4–FGF1 treatment. Upper pictures : without BMP4–FGF1 treatment; lower pictures : with BMP4–FGF1 treatment. The white scale bar represents 100 μm. E , relative mRNA expression levels of BMP4–FGF1 signaling and differentiation-related genes. n = 6–9. The mean mRNA expression level of control 3T3-L1 cells without BMP4–FGF1 treatment was set to 1. Gray : control cells without BMP4–FGF1 treatment; red : control cells with BMP4–FGF1 treatment; white : Ext1 +/− cells without BMP4–FGF1 treatment; blue : Ext1 +/− cells with BMP4–FGF1 treatment. ∗Comparison of gray and red ; † Comparison of gray and white ; # Comparison of white and blue . ∗ ,# p

    Techniques Used: Expressing

    31) Product Images from "CREG1 heterozygous mice are susceptible to high fat diet-induced obesity and insulin resistance"

    Article Title: CREG1 heterozygous mice are susceptible to high fat diet-induced obesity and insulin resistance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0176873

    Depletion of CREG1 expression enhances inflammatory responses through the NF-κB pathway in 3T3-L1 adipocytes. ( A ): Knockdown of CREG1 by siRNA (siCreg1) in differentiated 3T3-L1 adipocytes were verified by Western blot (left panel) (n = 3). Beta-actin was used as loading control. The right panel showed the quantification of CREG1 relative to β-actin (n = 3 from three independent experiments). ( B ): Relative mRNA expression of TNF-α, IL-6 and MCP-1 in siCreg1 or si-Scramble treated 3T3-L1 cells ± 10 ng/ml LPS (n = 3). ( C ): Representative Western blots showed the effect of siCreg1 on pro-inflammatory signaling pathways (n = 3 replicated 3 times). The right panel showed the quantification of CREG1/actin, p-IκBα/IκBα and p-NF-κB/Lamin B. Values are presented as means ± SEM. ns, no significance. * P
    Figure Legend Snippet: Depletion of CREG1 expression enhances inflammatory responses through the NF-κB pathway in 3T3-L1 adipocytes. ( A ): Knockdown of CREG1 by siRNA (siCreg1) in differentiated 3T3-L1 adipocytes were verified by Western blot (left panel) (n = 3). Beta-actin was used as loading control. The right panel showed the quantification of CREG1 relative to β-actin (n = 3 from three independent experiments). ( B ): Relative mRNA expression of TNF-α, IL-6 and MCP-1 in siCreg1 or si-Scramble treated 3T3-L1 cells ± 10 ng/ml LPS (n = 3). ( C ): Representative Western blots showed the effect of siCreg1 on pro-inflammatory signaling pathways (n = 3 replicated 3 times). The right panel showed the quantification of CREG1/actin, p-IκBα/IκBα and p-NF-κB/Lamin B. Values are presented as means ± SEM. ns, no significance. * P

    Techniques Used: Expressing, Western Blot

    32) Product Images from "SFMBT2-Mediated Infiltration of Preadipocytes and TAMs in Prostate Cancer"

    Article Title: SFMBT2-Mediated Infiltration of Preadipocytes and TAMs in Prostate Cancer

    Journal: Cancers

    doi: 10.3390/cancers12092718

    SFMBT2 knockdown LNCaP cells induce the infiltration of preadipocytes and TAMs in the xenograft model. ( A ) Schematic diagram of the xenograft experiment. LNCaP cells stably transfected with control (shCont) or SFMBT2 shRNA (shSFMBT2) were implanted subcutaneously into the flank of nude mice. After 4 weeks, stably RFP-expressing 3T3-L1 cells were injected into the tail vein of the same mouse. One week later, the tumor was harvested, fixed, sectioned, and immunostained with anti-RFP, anti-Pref-1, and anti-CD29 antibodies. ( B ) Increased number of infiltrated RFP-expressing 3T3-L1 preadipocytes in tumors. Representative images are shown. Nuclei were identified using DAPI staining. Scale bar, 25 μm. RFP positive cells were counted. ( C ) RFP expressing cells are Pref-1 and CD29 positive in tumors. Representative images are shown. Scale bar, 25 μm. ( D ) Increased number of infiltrated TAMs immunostained with anti-CD163 and anti-CD206 antibodies in tumors. Representative images are shown. Nuclei were identified using DAPI staining. Scale bar, 25 μm. CD163 or CD206 positive cells were counted. All data represent mean ± S.E.M. Significance values were * p ≤ 0.05 and ** p ≤ 0.01.
    Figure Legend Snippet: SFMBT2 knockdown LNCaP cells induce the infiltration of preadipocytes and TAMs in the xenograft model. ( A ) Schematic diagram of the xenograft experiment. LNCaP cells stably transfected with control (shCont) or SFMBT2 shRNA (shSFMBT2) were implanted subcutaneously into the flank of nude mice. After 4 weeks, stably RFP-expressing 3T3-L1 cells were injected into the tail vein of the same mouse. One week later, the tumor was harvested, fixed, sectioned, and immunostained with anti-RFP, anti-Pref-1, and anti-CD29 antibodies. ( B ) Increased number of infiltrated RFP-expressing 3T3-L1 preadipocytes in tumors. Representative images are shown. Nuclei were identified using DAPI staining. Scale bar, 25 μm. RFP positive cells were counted. ( C ) RFP expressing cells are Pref-1 and CD29 positive in tumors. Representative images are shown. Scale bar, 25 μm. ( D ) Increased number of infiltrated TAMs immunostained with anti-CD163 and anti-CD206 antibodies in tumors. Representative images are shown. Nuclei were identified using DAPI staining. Scale bar, 25 μm. CD163 or CD206 positive cells were counted. All data represent mean ± S.E.M. Significance values were * p ≤ 0.05 and ** p ≤ 0.01.

    Techniques Used: Stable Transfection, Transfection, shRNA, Mouse Assay, Expressing, Injection, Staining

    33) Product Images from "Metabolic Regulation of Transcription Through Compartmentalized NAD+ Biosynthesis"

    Article Title: Metabolic Regulation of Transcription Through Compartmentalized NAD+ Biosynthesis

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

    doi: 10.1126/science.aan5780

    NMNAT-1 regulates PARP-1 activity and adipocyte differentiation. (A) Schematic representation of NAD + biosynthesis by NMNATs and their subcellular localization. (B) Western blot showing the levels of PAR upon shRNA-mediated knockdown (KD) of Nmnat1 during the early phase of adipogenesis in 3T3-L1 cells. PAR levels (primarily automodification of PARP-1) represent the enzymatic activity of PARP-1. Blots of NMNAT-1, PARP-1, and SIRT1 are shown for comparison. (C and D) Accumulation of lipid droplets at 4 days (C) and 8 days (D) of differentiation after knockdown (KD) of Nmnat1 or Parp1 in 3T3-L1 cells. Lipids were stained using BODIPY 493/503 (green, C) or Oil red O (red, D) and nuclei were stained using TO-PRO-3 (blue, C).
    Figure Legend Snippet: NMNAT-1 regulates PARP-1 activity and adipocyte differentiation. (A) Schematic representation of NAD + biosynthesis by NMNATs and their subcellular localization. (B) Western blot showing the levels of PAR upon shRNA-mediated knockdown (KD) of Nmnat1 during the early phase of adipogenesis in 3T3-L1 cells. PAR levels (primarily automodification of PARP-1) represent the enzymatic activity of PARP-1. Blots of NMNAT-1, PARP-1, and SIRT1 are shown for comparison. (C and D) Accumulation of lipid droplets at 4 days (C) and 8 days (D) of differentiation after knockdown (KD) of Nmnat1 or Parp1 in 3T3-L1 cells. Lipids were stained using BODIPY 493/503 (green, C) or Oil red O (red, D) and nuclei were stained using TO-PRO-3 (blue, C).

    Techniques Used: Activity Assay, Western Blot, shRNA, Staining

    NMNAT-2 is a sensor of enhanced glucose metabolism during the early phase of adipogenesis. (A) Schematic representation of the potential role of NMNAT-1 and NMNAT-2 during adipocyte differentiation. (B) Nmnat2 knockdown (KD) does not affect the expression of genes involved in glucose metabolism after 8 hours of differentiation, as determined by RT-qPCR. None of the minor differences are significant (Student’s t-test; p > 0.05). (C) Nmnat2 knockdown (KD) alters glucose flux during the differentiation of 3T3-L1 cells. Mass isotopomer analysis of m+2 citrate in cells ± Nmnat2 knockdown (KD). Asterisks indicate significant differences from the corresponding control (Student’s t-test; * p
    Figure Legend Snippet: NMNAT-2 is a sensor of enhanced glucose metabolism during the early phase of adipogenesis. (A) Schematic representation of the potential role of NMNAT-1 and NMNAT-2 during adipocyte differentiation. (B) Nmnat2 knockdown (KD) does not affect the expression of genes involved in glucose metabolism after 8 hours of differentiation, as determined by RT-qPCR. None of the minor differences are significant (Student’s t-test; p > 0.05). (C) Nmnat2 knockdown (KD) alters glucose flux during the differentiation of 3T3-L1 cells. Mass isotopomer analysis of m+2 citrate in cells ± Nmnat2 knockdown (KD). Asterisks indicate significant differences from the corresponding control (Student’s t-test; * p

    Techniques Used: Expressing, Quantitative RT-PCR

    Nuclear NAD + levels are regulated through compartmentalized biosynthesis. (A) The levels of total intracellular NAD + (enzyme-linked NAD + assay), PAR (Western blot), and Nmnat2 mRNA (RT-qPCR) were determined at the indicated differentiation time points in 3T3-L1 cells. (B) Detection of nuclear (Nuc) and cytoplasmic (Cyto) NAD + levels in 3T3-L1 cells using a cpVenus-based NAD + biosensor. Representative images of NAD + sensor fluorescence during the early phase of differentiation are shown. (C) Changes in subcellular NAD + levels during the early phase of differentiation of 3T3-L1 cells. NAD + levels were calculated from sensor (488/405 nm) /control (488/405 nm) fluorescence ratios determined by flow cytometry using a standard curve. Each bar represents the mean ± SEM, n = 7. Bars marked with asterisks are significantly different from the undifferentiated (0 hr) control (ANOVA; ** p
    Figure Legend Snippet: Nuclear NAD + levels are regulated through compartmentalized biosynthesis. (A) The levels of total intracellular NAD + (enzyme-linked NAD + assay), PAR (Western blot), and Nmnat2 mRNA (RT-qPCR) were determined at the indicated differentiation time points in 3T3-L1 cells. (B) Detection of nuclear (Nuc) and cytoplasmic (Cyto) NAD + levels in 3T3-L1 cells using a cpVenus-based NAD + biosensor. Representative images of NAD + sensor fluorescence during the early phase of differentiation are shown. (C) Changes in subcellular NAD + levels during the early phase of differentiation of 3T3-L1 cells. NAD + levels were calculated from sensor (488/405 nm) /control (488/405 nm) fluorescence ratios determined by flow cytometry using a standard curve. Each bar represents the mean ± SEM, n = 7. Bars marked with asterisks are significantly different from the undifferentiated (0 hr) control (ANOVA; ** p

    Techniques Used: Western Blot, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Cytometry

    Model for the coordination of transcription and glucose metabolism during adipocyte differentiation through compartmentalized NAD + biosynthesis. In undifferentiated 3T3-L1 cells, NMN is used mostly by NMNAT-1 to synthesize nuclear NAD + , which supports PARP-1 activity. Active PARP-1 ADP-ribosylates the adipogenic transcription factor C/EBP , which inhibits its chromatin binding and transcriptional activities, preventing differentiation in the absence of an adipogenic signal. During differentiation, the expression of genes involved in glucose metabolism increases, leading to a rapid induction of glucose flux in 3T3-L1 cells. Concurrently, NMNAT-2 is rapidly induced to support the high local NAD + demands caused by enhanced glucose metabolism, thereby limiting NMN availability in the nucleus for NMNAT-1 to synthesize nuclear NAD + . Reduced nuclear NAD + concentrations lead to reduced PARP-1 activity, allowing C/EBP to initiate the adipogenic transcription program. Competition for the NAD + precursor, NMN, between the nuclear and cytoplasmic NMNATs results in changes in nuclear NAD + levels, allowing cells to coordinate glucose metabolism and transcription.
    Figure Legend Snippet: Model for the coordination of transcription and glucose metabolism during adipocyte differentiation through compartmentalized NAD + biosynthesis. In undifferentiated 3T3-L1 cells, NMN is used mostly by NMNAT-1 to synthesize nuclear NAD + , which supports PARP-1 activity. Active PARP-1 ADP-ribosylates the adipogenic transcription factor C/EBP , which inhibits its chromatin binding and transcriptional activities, preventing differentiation in the absence of an adipogenic signal. During differentiation, the expression of genes involved in glucose metabolism increases, leading to a rapid induction of glucose flux in 3T3-L1 cells. Concurrently, NMNAT-2 is rapidly induced to support the high local NAD + demands caused by enhanced glucose metabolism, thereby limiting NMN availability in the nucleus for NMNAT-1 to synthesize nuclear NAD + . Reduced nuclear NAD + concentrations lead to reduced PARP-1 activity, allowing C/EBP to initiate the adipogenic transcription program. Competition for the NAD + precursor, NMN, between the nuclear and cytoplasmic NMNATs results in changes in nuclear NAD + levels, allowing cells to coordinate glucose metabolism and transcription.

    Techniques Used: Activity Assay, Binding Assay, Expressing

    34) Product Images from "Zinc-?2-glycoprotein, a lipid mobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia"

    Article Title: Zinc-?2-glycoprotein, a lipid mobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia

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

    doi: 10.1073/pnas.0308647100

    ZAG gene expression in 3T3-L1 adipocytes by real-time RT-PCR. ( A ) Expression during differentiation of 3T3-L1 cells into adipocytes. Data are means ± SEM, relative to day 0 ( n = 3). ( B ) Effects of BRL 37344 or dexamethasone (DEX) on ZAG mRNA levels in 3T3-L1 adipocytes. Cells were taken at day 8 after induction of differentiation and incubated for 24 h with BRL 37344 (1μM) or dexamethasone (20 nM). ZAG mRNA levels were normalized to β-actin. Data are means ± SEM ( n = 6); changes are relative to controls. * , P
    Figure Legend Snippet: ZAG gene expression in 3T3-L1 adipocytes by real-time RT-PCR. ( A ) Expression during differentiation of 3T3-L1 cells into adipocytes. Data are means ± SEM, relative to day 0 ( n = 3). ( B ) Effects of BRL 37344 or dexamethasone (DEX) on ZAG mRNA levels in 3T3-L1 adipocytes. Cells were taken at day 8 after induction of differentiation and incubated for 24 h with BRL 37344 (1μM) or dexamethasone (20 nM). ZAG mRNA levels were normalized to β-actin. Data are means ± SEM ( n = 6); changes are relative to controls. * , P

    Techniques Used: Expressing, Quantitative RT-PCR, Incubation

    ZAG gene expression in mouse tissues and adipocytes examined by RT-PCR. ( A ) Tissue specificity of expression. ( B ) Expression in fat depots. ( C ) Expression in mature adipocytes and stromal-vascular cells (isolated by collagenase digestion) and 3T3-L1 adipocytes. The 3T3-L1 cells were taken at days 3 and 6 after inducing differentiation. HPRT, hypoxanthine-phosphoribosyl transferase; e-WAT, epididymal WAT; peri, perirenal; sub, s.c.; sk, skeletal; S-V, stromal vascular.
    Figure Legend Snippet: ZAG gene expression in mouse tissues and adipocytes examined by RT-PCR. ( A ) Tissue specificity of expression. ( B ) Expression in fat depots. ( C ) Expression in mature adipocytes and stromal-vascular cells (isolated by collagenase digestion) and 3T3-L1 adipocytes. The 3T3-L1 cells were taken at days 3 and 6 after inducing differentiation. HPRT, hypoxanthine-phosphoribosyl transferase; e-WAT, epididymal WAT; peri, perirenal; sub, s.c.; sk, skeletal; S-V, stromal vascular.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

    35) Product Images from "Anandamide-derived Prostamide F2α"

    Article Title: Anandamide-derived Prostamide F2α

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.489906

    Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α
    Figure Legend Snippet: Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A , mRNA levels of Pparg and Cebpa were determined by QPCR in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO ( D ), bimatoprost, PGF 2α

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ
    Figure Legend Snippet: Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B , mRNA levels of Pparg , Cebpa , and Ptgs2 were determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 μ m bimatoprost, 5 μ

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

    Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ
    Figure Legend Snippet: Effect of MEK inhibition on prostamide antiadipogenic activity. A and D , mRNA levels of Pparg , Cebpa , or Ptgs2 were determined by QPCR in differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 μ m bimatoprost ( Bim .), 10 μ

    Techniques Used: Inhibition, Activity Assay, Real-time Polymerase Chain Reaction

    Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence
    Figure Legend Snippet: Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A , quantification (mean ± S.E.) of PGF 2α EA levels by LC-APCI-MS in undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone ( left panel ) or in the presence

    Techniques Used: Mass Spectrometry

    36) Product Images from "Myonectin inhibits adipogenesis in 3T3-L1 preadipocytes by regulating p38 MAPK pathway"

    Article Title: Myonectin inhibits adipogenesis in 3T3-L1 preadipocytes by regulating p38 MAPK pathway

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2021.54.2.262

    Suppression of adipogenic differentiation by treatment with myonectin. (A) Effects of myonectin on cell viability. The viability of untreated control cells (Non) was defined as 100%. (B) Effects of myonectin on the differentiation of 3T3-L1 cells. Differentiation of con-fluent 3T3-L1 cells was induced by the MDI cocktail (0.5 mM IBMX, 1 μM Dexamethasone, and 10 μg/ml insulin). The 3T3-L1 cells were treated with different concentrations (0.05-2 μg/ml) of myonectin during differentiation. On day 10 of differentiation, the fully differentiated adipocytes were fixed and stained with oil red O solution for visualization of the lipid accumulation. (C-F) mRNA levels of adipogenic genes in mature 3T3-L1 adipocytes. Each gene was normalized to Rpl32. All quantitative data are the means ± SD (n = 6). *P
    Figure Legend Snippet: Suppression of adipogenic differentiation by treatment with myonectin. (A) Effects of myonectin on cell viability. The viability of untreated control cells (Non) was defined as 100%. (B) Effects of myonectin on the differentiation of 3T3-L1 cells. Differentiation of con-fluent 3T3-L1 cells was induced by the MDI cocktail (0.5 mM IBMX, 1 μM Dexamethasone, and 10 μg/ml insulin). The 3T3-L1 cells were treated with different concentrations (0.05-2 μg/ml) of myonectin during differentiation. On day 10 of differentiation, the fully differentiated adipocytes were fixed and stained with oil red O solution for visualization of the lipid accumulation. (C-F) mRNA levels of adipogenic genes in mature 3T3-L1 adipocytes. Each gene was normalized to Rpl32. All quantitative data are the means ± SD (n = 6). *P

    Techniques Used: Staining

    37) Product Images from "In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors"

    Article Title: In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    doi: 10.1080/14756366.2021.1971976

    Cytotoxicity assay of compounds 5e , 5 g , 5 h , and 5 s on 3T3-L1 cells and HepG2 cells.
    Figure Legend Snippet: Cytotoxicity assay of compounds 5e , 5 g , 5 h , and 5 s on 3T3-L1 cells and HepG2 cells.

    Techniques Used: Cytotoxicity Assay

    38) Product Images from "Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling"

    Article Title: Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling

    Journal: Scientific Reports

    doi: 10.1038/srep42104

    Rosiglitazone Induces Varied Adipogenic Activities Based on Cell Culture Plastic. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) using various tissue culture plates. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone cultured in Greiner Bio-One CELLSTAR™, Brandtech cellGrade™, and Labnet International Krystal™ 2000 tissue culture plates for ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for rosiglitazone cultured in the tissue culture plates described above for ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for rosiglitazone cultured in the tissue culture plates described above for ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Fold induction of triglyceride accumulative response over vehicle control for rosiglitazone cultured in the cell culture plastics described above for ATCC 3T3-L1 cells ( J ), Zenbio 3T3-L1 cells ( K ), and OP9 cells ( L ). Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p
    Figure Legend Snippet: Rosiglitazone Induces Varied Adipogenic Activities Based on Cell Culture Plastic. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) using various tissue culture plates. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone cultured in Greiner Bio-One CELLSTAR™, Brandtech cellGrade™, and Labnet International Krystal™ 2000 tissue culture plates for ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for rosiglitazone cultured in the tissue culture plates described above for ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for rosiglitazone cultured in the tissue culture plates described above for ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Fold induction of triglyceride accumulative response over vehicle control for rosiglitazone cultured in the cell culture plastics described above for ATCC 3T3-L1 cells ( J ), Zenbio 3T3-L1 cells ( K ), and OP9 cells ( L ). Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p

    Techniques Used: Cell Culture, Staining, Concentration Assay

    Bisphenol A and Analogs Induce Varied Adipogenic Activities Between Cell Lines. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with four bisphenol A analogs. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for bisphenol A (BPA), tetrabrominated bisphenol A (TBBPA), tetrachlorinated bisphenol A (TCBPA), and hexafluorinated bisphenol A (BPAF) in ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Triglyceride accumulation responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p
    Figure Legend Snippet: Bisphenol A and Analogs Induce Varied Adipogenic Activities Between Cell Lines. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with four bisphenol A analogs. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for bisphenol A (BPA), tetrabrominated bisphenol A (TBBPA), tetrachlorinated bisphenol A (TCBPA), and hexafluorinated bisphenol A (BPAF) in ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Triglyceride accumulation responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p

    Techniques Used: Staining, Activity Assay, Concentration Assay

    Mechanistic Receptor Controls Induce Varied Adipogenic Activities Between Cell Lines. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with mechanistic receptor control ligands. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone (RSG, PPARγ agonist), GW3965 (liver X receptor, LXR, agonist), dexamethasone (glucocorticoid receptor, GR, agonist), 1–850 (thyroid receptor, TR, antagonist), flutamide (androgen receptor, AR, antagonist), and LG100268 (retinoid X receptor, RXR, agonist) in ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p
    Figure Legend Snippet: Mechanistic Receptor Controls Induce Varied Adipogenic Activities Between Cell Lines. ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with mechanistic receptor control ligands. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone (RSG, PPARγ agonist), GW3965 (liver X receptor, LXR, agonist), dexamethasone (glucocorticoid receptor, GR, agonist), 1–850 (thyroid receptor, TR, antagonist), flutamide (androgen receptor, AR, antagonist), and LG100268 (retinoid X receptor, RXR, agonist) in ATCC 3T3-L1 cells ( A ), Zenbio 3T3-L1 cells ( B ), and OP9 cells ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells ( D ), Zenbio 3T3-L1 cells ( E ), and OP9 cells ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells ( G ), Zenbio 3T3-L1 cells ( H ), and OP9 cells ( I ). Responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p

    Techniques Used: Staining, Activity Assay, Concentration Assay

    39) Product Images from "Inhibition of the Monocarboxylate Transporter 1 (MCT1) Promotes 3T3-L1 Adipocyte Proliferation and Enhances Insulin Sensitivity"

    Article Title: Inhibition of the Monocarboxylate Transporter 1 (MCT1) Promotes 3T3-L1 Adipocyte Proliferation and Enhances Insulin Sensitivity

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23031901

    Insulin responsiveness. Glucose uptake in differentiated 3T3-L1 (WT) cells. ( A ) Glucose uptake in 3T3-L1 cells treated with 1 µM AZD3965 for 0 h (solid bars) or 24 h, 48 h, and 72 h (striped bars) were treated with (red bars) or without (blue bars) insulin for 30 min (**** p
    Figure Legend Snippet: Insulin responsiveness. Glucose uptake in differentiated 3T3-L1 (WT) cells. ( A ) Glucose uptake in 3T3-L1 cells treated with 1 µM AZD3965 for 0 h (solid bars) or 24 h, 48 h, and 72 h (striped bars) were treated with (red bars) or without (blue bars) insulin for 30 min (**** p

    Techniques Used:

    40) Product Images from "Salvianolic acid-B improves fat graft survival by promoting proliferation and adipogenesis"

    Article Title: Salvianolic acid-B improves fat graft survival by promoting proliferation and adipogenesis

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-021-02575-4

    Effect of Sal-B on cell viability in 3T3-L1 cells and ADSCs. ( a ) Chemical structure of Salvianolic acid B (Sal-B). ( b ) Cell viability was determined with CCK-8 reagent after Sal-B treatment for 72 h (left: 3T3-L1, right: ADSCs). ( c ) EdU (green) proliferation assay was performed at 24 h after the addition of 10, 50 or 100 μmol/L Sal-B (PBS was added instead in the control group). Hoechst33342 (blue) staining of nuclei. Bar = 200 μm. The data represent the mean ± SD. * P
    Figure Legend Snippet: Effect of Sal-B on cell viability in 3T3-L1 cells and ADSCs. ( a ) Chemical structure of Salvianolic acid B (Sal-B). ( b ) Cell viability was determined with CCK-8 reagent after Sal-B treatment for 72 h (left: 3T3-L1, right: ADSCs). ( c ) EdU (green) proliferation assay was performed at 24 h after the addition of 10, 50 or 100 μmol/L Sal-B (PBS was added instead in the control group). Hoechst33342 (blue) staining of nuclei. Bar = 200 μm. The data represent the mean ± SD. * P

    Techniques Used: CCK-8 Assay, Proliferation Assay, Staining

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    ATCC 3t3 l1 preadipocytes
    Cytotoxic effect of LM on <t>3T3-L1</t> preadipocytes. (a) LM effect on <t>3T3-L1</t> 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.
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    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

    M. canettii infection of 3T3-L1 mature adipocytes. (A) Confocal images of 3T3-L1 mature adipocytes infected with M. canettii . Adipocytes were infected at MOI 10:1 with M. canettii mCherry for 4 h. At day 3 post-inoculation, extracellular mycobacteria were washed off, infected cells were fixed in 4% formaldehyde and the lipid droplets of adipocytes were stained using BODIPY 493/503. Confocal images showed intracellular location of M. canettii mCherry and 30% of intracellular bacilli were next to lipid bodies (White squares). (B) Electron microscopy observations of 3T3-L1 mature adipocytes infected with M. canettii . Adipocytes were infected at MOI 5:1 with M. canettii for 4 h. At day 3 post-inoculation, the supernatant was removed and infected cells were fixed for electron microscopy. M. canettii organisms were inside adipocytes in close contact with adipocytes LBs as indicated by the red arrow a nd accumulated ILIs (upper panel). Pre-adipocytes that failed to reach full differentiation were infected also with M. canettii (lower panel).

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mycobacterium canettii Infection of Adipose Tissues

    doi: 10.3389/fcimb.2017.00189

    Figure Lengend Snippet: M. canettii infection of 3T3-L1 mature adipocytes. (A) Confocal images of 3T3-L1 mature adipocytes infected with M. canettii . Adipocytes were infected at MOI 10:1 with M. canettii mCherry for 4 h. At day 3 post-inoculation, extracellular mycobacteria were washed off, infected cells were fixed in 4% formaldehyde and the lipid droplets of adipocytes were stained using BODIPY 493/503. Confocal images showed intracellular location of M. canettii mCherry and 30% of intracellular bacilli were next to lipid bodies (White squares). (B) Electron microscopy observations of 3T3-L1 mature adipocytes infected with M. canettii . Adipocytes were infected at MOI 5:1 with M. canettii for 4 h. At day 3 post-inoculation, the supernatant was removed and infected cells were fixed for electron microscopy. M. canettii organisms were inside adipocytes in close contact with adipocytes LBs as indicated by the red arrow a nd accumulated ILIs (upper panel). Pre-adipocytes that failed to reach full differentiation were infected also with M. canettii (lower panel).

    Article Snippet: 3T3-L1 cell lines Murine embryonic fibroblasts 3T3-L1 (ATCC, lot 62158491) were cultured in DMEM (Invitrogen, France) supplemented with 10% heat-inactivated FBS at 37°C under a 5% CO2 atmosphere.

    Techniques: Infection, Staining, Electron Microscopy

    Intracellular kinetic of M. canettii CIPT 140010059 and M. tuberculosis H37Rv in 3T3-L1 pre-adipocytes and adipocytes during 12-day experiments . Cells were infected at MOI 1:1 with M. canettii or M. tuberculosis for 4 h. The number of bacterial colony forming units (CFU) was determined at the indicated time points (0, 3, 7, and 12 day p.i.). A similar intracellular kinetic was observed for M. canettii and M. tuberculosis inside pre-adipocytes. However, in mature adipocytes M. tuberculosis stopped its replication between day 3 and 12 day p.i. while M. canettii continued to replicate (arrow). Data shown are the mean ± standard deviation of three independent experiments.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mycobacterium canettii Infection of Adipose Tissues

    doi: 10.3389/fcimb.2017.00189

    Figure Lengend Snippet: Intracellular kinetic of M. canettii CIPT 140010059 and M. tuberculosis H37Rv in 3T3-L1 pre-adipocytes and adipocytes during 12-day experiments . Cells were infected at MOI 1:1 with M. canettii or M. tuberculosis for 4 h. The number of bacterial colony forming units (CFU) was determined at the indicated time points (0, 3, 7, and 12 day p.i.). A similar intracellular kinetic was observed for M. canettii and M. tuberculosis inside pre-adipocytes. However, in mature adipocytes M. tuberculosis stopped its replication between day 3 and 12 day p.i. while M. canettii continued to replicate (arrow). Data shown are the mean ± standard deviation of three independent experiments.

    Article Snippet: 3T3-L1 cell lines Murine embryonic fibroblasts 3T3-L1 (ATCC, lot 62158491) were cultured in DMEM (Invitrogen, France) supplemented with 10% heat-inactivated FBS at 37°C under a 5% CO2 atmosphere.

    Techniques: Infection, Standard Deviation

    Genes involved in adipogenesis and analyzed in this study. During the course of differentiation, two waves of transcription factors direct preadipocytes to change into adipocytes. In 3T3-L1 cells, the first wave is induced by a cocktail of IBMX, dexamethasone, and insulin (MDI), causing enhanced transient transcription of CAAT/enhancer binding proteins, Cebpβ and Cebpδ, followed by expression of sterol-responsive element binding protein 1a ( Srebf1a ). These transcription factors induce a second wave of transcription factors ( Pparγ2 and Cebpα ). PPARγ2 will form a heterodimer with retinoic x receptor (RXR), causing expression of PPARγ2 targets such as fatty acid binding protein 4 ( Fabp4 ), lipoprotein lipase ( Lpl ), glucose transporter type 4 ( Slc2a4 ), and adiponectin ( Adipoq ). Mature adipocytes will differentially express genes involved in glucose homeostasis compared to preadipocytes (e.g., increases in glucose 6 phosphatase catalytic subunit ( G6pc ), insulin growth factor 1 ( Igf1 ), leptin ( Lep ), and insulin receptor ( Insr ), and a decrease in insulin growth factor receptor ( Igfr )). 14 , 40 − 42

    Journal: Environmental Science & Technology

    Article Title: Transcriptional and Epigenetic Mechanisms Underlying Enhanced in Vitro Adipocyte Differentiation by the Brominated Flame Retardant BDE-47

    doi: 10.1021/es405524b

    Figure Lengend Snippet: Genes involved in adipogenesis and analyzed in this study. During the course of differentiation, two waves of transcription factors direct preadipocytes to change into adipocytes. In 3T3-L1 cells, the first wave is induced by a cocktail of IBMX, dexamethasone, and insulin (MDI), causing enhanced transient transcription of CAAT/enhancer binding proteins, Cebpβ and Cebpδ, followed by expression of sterol-responsive element binding protein 1a ( Srebf1a ). These transcription factors induce a second wave of transcription factors ( Pparγ2 and Cebpα ). PPARγ2 will form a heterodimer with retinoic x receptor (RXR), causing expression of PPARγ2 targets such as fatty acid binding protein 4 ( Fabp4 ), lipoprotein lipase ( Lpl ), glucose transporter type 4 ( Slc2a4 ), and adiponectin ( Adipoq ). Mature adipocytes will differentially express genes involved in glucose homeostasis compared to preadipocytes (e.g., increases in glucose 6 phosphatase catalytic subunit ( G6pc ), insulin growth factor 1 ( Igf1 ), leptin ( Lep ), and insulin receptor ( Insr ), and a decrease in insulin growth factor receptor ( Igfr )). 14 , 40 − 42

    Article Snippet: 3T3-L1 Cell Culture 3T3-L1 cells (ATCC, Manassas, VA) were maintained in DMEM (high glucose, 15 mM HEPES, and glutamax) (Gibco, The Netherlands), supplemented with 1× nonessential amino acids (Gibco, The Netherlands), 10% FCS (Sigma Aldrich, Germany), and penicillin/streptomycin (Gibco, The Netherlands).

    Techniques: Binding Assay, Expressing

    DEX induces the change in NCoR mRNA splicing observed during 3T3-L1 cells differentiation. Cells were differentiated with various combinations of the individual factors within the tripartite differentiation cocktail. Means ± S.E.M. (n = 3 for SMRT Exon 40 and NCoR Exon 28, and n = 5 for NCoR Exon 37) are presented. p

    Journal: Molecular and cellular endocrinology

    Article Title: Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling

    doi: 10.1016/j.mce.2015.06.036

    Figure Lengend Snippet: DEX induces the change in NCoR mRNA splicing observed during 3T3-L1 cells differentiation. Cells were differentiated with various combinations of the individual factors within the tripartite differentiation cocktail. Means ± S.E.M. (n = 3 for SMRT Exon 40 and NCoR Exon 28, and n = 5 for NCoR Exon 37) are presented. p

    Article Snippet: The 3T3-L1 adipocyte model is used to mimic in vivo adipo-genesis by the treatment of undifferentiated 3T3-L1 cells, which display a fibroblast-like preadipocyte phenotype, with a “differentiation cocktail” that induces the cells to differentiate into a mature, lipid-accumulating, adipocyte-like phenotype ( ).

    Techniques: