Journal: Scientific Reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Release of glycerol and lactate to the extracellular medium in 3T3-L1 adipocytes over-expressing Dlk or Notch genes. Relative levels of glycerol released to the extracellular medium in response to isoproterenol from Dlk1 or Dlk2 genes ( A ), and Notch1 , 2 , 3 or 4 genes (B) over-expressing adipocytes. ( C ) Representative microscopy images (400X magnification) of non-transfected (L1C) and transfected 3T3-L1 adipocytes (Empty vectors V1, V2, V3 and V4, and their corresponding over-expressing transfectant) under study. The size of their lipid droplets is showed. Scale bar (80 μm) is shown. Relative levels of lactate in the culture supernatant of differentiated non-transfected 3T3-L1 cells ( D ), Dlk1 or Dlk2 genes over-expressing adipocytes ( E ), and each of the Notch genes over-expressing adipocytes ( F ). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The cell lines used were: 3T3-L1 (L1; ATCC CCL-92.1); HEK 293 T/17 (ATCC CRL-11268); and Balb/c14 (Balb/c 3T3 sub-clones negative for Dlk1 expression .

Techniques: Expressing, Microscopy, Transfection, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific Reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Effects of stable over-expression of each one of the Notch genes in 3T3-L1 adipocyte browning. ( A ) Representative microscopy images (400X magnification) of 3T3-L1 adipocytes (L1D) seven days after standard adipogenic induction (48 hours with IBMX and dexamethasone, and 5 days with insulin, see Methods) and non-treated 3T3-L1 cells (L1C). Scale bar (250 μm) is shown. ( B ) qRT-PCR mRNA expression analysis of the brown adipocyte markers Ucp1 , Pgc1a , Gyk , Prdm16 , Cidea and Sirt1 in seven-day-differentiated 3T3-L1 cells. qRT-PCR analysis of the relative mRNA expression levels of Ucp1 , Pgc1a , Gyk , Prdm16 , Cidea and Sirt1 markers in seven-day differentiated Notch1 gene transfectant (L1-N1D) ( C ), Notch2 gene transfectant (L1-N2D) ( D ), Notch3 gene transfectant (L1-N3D) ( E ), Notch4 gene transfectant (L1-N4D) ( F ). Data from qRT-PCR assays were previously normalized to P0 mRNA expression levels. qPCR analysis of mitochondrial CytB DNA amplification (related to genomic ApoB DNA amplification, see Methods) in seven-day differentiated 3T3-L1 cells over-expressing Notch1 gene ( G ) and Notch2 , Notch3 or Notch4 genes ( H ). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of the Student’s t-tests performed is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The cell lines used were: 3T3-L1 (L1; ATCC CCL-92.1); HEK 293 T/17 (ATCC CRL-11268); and Balb/c14 (Balb/c 3T3 sub-clones negative for Dlk1 expression .

Techniques: Over Expression, Microscopy, Quantitative RT-PCR, Expressing, Transfection, Real-time Polymerase Chain Reaction, Amplification, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific Reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: NOTCH activation and signaling in 3T3-L1 cells stably over-expressing each one of the four NOTCH receptors. ( A ) qRT-PCR analysis of the relative Hes1 and Hey1 mRNA expression levels in the stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). ( B ) NOTCH transcriptional activity, as measured by gene reporter luciferase assays, in these four Notch genes stable transfectants. ( C ) qRT-PCR analysis of the relative individual Notch mRNA expression levels in stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). The relative luciferase activities were always normalized with renilla values and referred to those of control cells. Data in all qRT-PCR assays were normalized to P0 mRNA expression levels. The fold activation or inhibition in all assays is measured relative to the empty vector control, set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of Student’s t-tests results is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The cell lines used were: 3T3-L1 (L1; ATCC CCL-92.1); HEK 293 T/17 (ATCC CRL-11268); and Balb/c14 (Balb/c 3T3 sub-clones negative for Dlk1 expression .

Techniques: Activation Assay, Stable Transfection, Expressing, Quantitative RT-PCR, Transfection, Activity Assay, Luciferase, Inhibition, Plasmid Preparation

Journal: Scientific Reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Feedback modulation among Notch and Dlk gene expression in 3T3-L1 preadipocytes. ( A ) qRT-PCR analysis of the relative individual Dlk ( B ) mRNA expression levels in the stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). ( B ) qRT-PCR analysis of the relative Hes1 and Dlk mRNA expression levels in the stable Hes1 gene transfectant (L1-H1). ( C ) qRT-PCR analysis of the relative Hes1 and Hey1 mRNA expression levels in the stable Dlk1 gene transfectant (L1-DLK1) and the stable Dlk2 gene transfectant (L1-DLK2). qRT-PCR analysis of the relative Notch ( D ) and Dlk ( E ) mRNA expression levels in the stable Dlk1 gene transfectant (L1-DLK1), and the stable Dlk2 gene transfectant (L1-DLK2). In all qRT-PCR assays, data were normalized to P0 mRNA expression levels. The fold activation or inhibition in all assays was measured relative to the empty vector, set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of Student’s t-tests results is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The cell lines used were: 3T3-L1 (L1; ATCC CCL-92.1); HEK 293 T/17 (ATCC CRL-11268); and Balb/c14 (Balb/c 3T3 sub-clones negative for Dlk1 expression .

Techniques: Expressing, Quantitative RT-PCR, Transfection, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific Reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Effects of stable over-expression of Dlk1 or Dlk2 genes in 3T3-L1 adipocyte browning. ( A ) qRT-PCR analysis of the relative mRNA expression levels of Ucp1 , Pgc1a , Gyk , Prdm16 , Cidea and Sirt1 markers in seven-day differentiated Dlk1 gene transfectant (L1-DLK1D) and Dlk2 gene transfectant (L1-DLK2D). Data from qRT-PCR assays were previously normalized to P0 mRNA expression levels. ( B ) qPCR analysis of mitochondrial CytB DNA amplification (related to genomic ApoB DNA amplification, see Methods) in seven-day differentiated 3T3-L1 cells over-expressing Dlk1 or Dlk2 genes. The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of the Student’s t-tests performed is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The cell lines used were: 3T3-L1 (L1; ATCC CCL-92.1); HEK 293 T/17 (ATCC CRL-11268); and Balb/c14 (Balb/c 3T3 sub-clones negative for Dlk1 expression .

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Real-time Polymerase Chain Reaction, Amplification, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific Reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Oxygen consumption rate (OCR) in 3T3-L1 adipocytes over-expressing Dlk and Notch genes. Analysis of the relative oxygen consumption rate (OCR) in non-transfected 3T3-L1 cells ( A ) and 3T3-L1 cells over-expressing Dlk1 or Dlk2 genes ( B ), and Notch1 ( C ), Notch2 ( D ), Notch3 ( E ) or Notch4 genes ( F ). The fold activation or inhibition was calculated relative to the time 0 of seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated at 120 minutes (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The cell lines used were: 3T3-L1 (L1; ATCC CCL-92.1); HEK 293 T/17 (ATCC CRL-11268); and Balb/c14 (Balb/c 3T3 sub-clones negative for Dlk1 expression .

Techniques: Expressing, Transfection, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific Reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: The stable over-expression of each one of the Notch genes on 3T3-L1 cells enhances adipogenesis. ( A ) qRT-PCR analysis of the relative mRNA expression levels of the adipocyte markers aP2 and Pparg in differentiated 3T3-L1 cells. ( B ) qRT-PCR analysis of the relative Notch and Hes1 mRNA expression levels in differentiated 3T3-L1 cells. Representative Western blots ( C ) and densitometric analysis ( D ) of each NOTCH receptor expression in 3T3-L1 adipocytes compared to non-differentiated cells. In the case of the Notch1 gene transfectant intracellular NOTCH1 (NICD1) and complete NOTCH1 protein signals are shown. In the case of the Notch2 gene transfectant, the intracellular NOTCH2 (NICD2) protein signal is shown. For the Notch3 gene transfectant, the complete and the intracellular NOTCH3 (NICD3) protein signals are shown. Finally, for the Notch 4 gene transfectant, the complete and the intracellular NOTCH4 (NICD4) protein signals are shown. ( E ) qRT-PCR analysis of the relative aP2 and Pparg mRNA expression levels in differentiated stable Notch1 gene transfectant (L1-N1D), stable Notch2 gene transfectant (L1-N2D), stable Notch3 gene transfectant (L1-N3D), and stable gene Notch4 transfectant (L1-N4D). Data from qRT-PCR assays were previously normalized to P0 ). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The cell lines used were: 3T3-L1 (L1; ATCC CCL-92.1); HEK 293 T/17 (ATCC CRL-11268); and Balb/c14 (Balb/c 3T3 sub-clones negative for Dlk1 expression .

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Activation Assay, Inhibition, Plasmid Preparation