Journal: Cells

Article Title: NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

doi: 10.3390/cells9092032

Figure Lengend Snippet: Primary and secondary antibodies used in Western blot analysis.

Article Snippet: Plasmid pC-N1 (N1S) contains the complete mouse Notch1 gene cDNA (ATCC clone: MBA-105) in sense orientation [ ].

Techniques: Western Blot

Journal: Cells

Article Title: NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

doi: 10.3390/cells9092032

Figure Lengend Snippet: NOTCH signaling in mesenchymal C3H10T1/2 cells stably overexpressing NOTCH receptors or the HES1 protein. ( A ) NOTCH receptors’ transcriptional activity, as measured by luciferase assays, in each of the four stably Notch -transfected pools. The relative luciferase activities were always normalized to renilla levels. ( B ) qRT-PCR analysis of the relative Hes1 and Hey1 mRNA transcription levels in each of the stably Notch -transfected pools. ( C ) qRT-PCR analysis of relative mRNA transcription levels of Notch genes in the stable Notch1 transfectant (N1S), the stable Notch2 transfectant (N2S), the stable Notch3 transfectant (N3S), and the stable Notch4 transfectant (N4S). ( D ) qRT-PCR analysis of Dlk1 (DELTA-like 1 homolog) and Dlk2 (DELTA-like 2 homolog) mRNA transcription levels in the stable Notch transfectants. ( E ) qRT-PCR analysis of the relative Hes1 and Dlk mRNA transcription levels in the stable Hes1 gene transfectant (H1S). All these assays were performed using non-differentiated cells. Data in qRT-PCR assays were normalized to P0 mRNA transcription levels. The fold activation or inhibition in all assays was measured relative to levels in non-differentiated empty-vector-transfected cells, set arbitrarily at 1 (horizontal black line or V). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of Student’s t -test results is indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Non-statistical significance is indicated by ns.

Article Snippet: Plasmid pC-N1 (N1S) contains the complete mouse Notch1 gene cDNA (ATCC clone: MBA-105) in sense orientation [ ].

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

Journal: Cells

Article Title: NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

doi: 10.3390/cells9092032

Figure Lengend Snippet: Stable overexpression of Notch and Dlk genes in multipotent C3H10T1/2 cells enhanced adipogenesis levels. Representative microscopic images of adipocytes from C3H10T1/2 cells transfected with Notch ( A ) or Dlk genes ( B ) showing their adipogenic levels (50× magnification images, scale bar 200 μm) after oil red O staining, all compared with the adipogenic levels of empty-vector transfected cells (VD). D: differentiated cells. qRT-PCR analysis of indicated adipogenic marker mRNA transcription levels in the differentiated stable Notch1 -overexpressing cells (N1SD) ( C ), the differentiated stable Notch2- overexpressing cells (N2SD) ( D ), the differentiated stable Notch3 -overexpressing cells (N3SD) ( E ), the differentiated stable Notch4- overexpressing cells (N4SD) ( F ), the differentiated stable Dlk1 -overexpressing cells (DLK1SD) ( G ), and the differentiated stable Dlk2 -overexpressing cells (DLK2SD) ( H ). Data from all qRT-PCR assays were normalized to P0 mRNA transcription levels. The fold activation or inhibition levels in qRT-PCR assays were calculated relative to the levels shown by seven-day differentiated empty-vector-transfected cells, which were set arbitrarily at 1 (horizontal black line). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t -test is indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Non-statistical significance is indicated by ns.

Article Snippet: Plasmid pC-N1 (N1S) contains the complete mouse Notch1 gene cDNA (ATCC clone: MBA-105) in sense orientation [ ].

Techniques: Over Expression, Transfection, Staining, Plasmid Preparation, Quantitative RT-PCR, Marker, Activation Assay, Inhibition

Journal: Cells

Article Title: NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

doi: 10.3390/cells9092032

Figure Lengend Snippet: Stable overexpression of Notch and Dlk genes in multipotent C3H10T1/2 cells modulated brown adipogenesis. ( A ) Representative microscopic images of adipocytes of C3H10T1/2 cells transfected with Notch ( A ) or Dlk genes ( B ), showing the size of their lipid droplets (400× magnification images, scale bar 30 μm) after oil red O staining, all of them compared with the size of lipid droplets of empty-vector-transfected differentiated cells (VD). qRT-PCR analysis of the indicated brown marker expression levels in the differentiated stably Notch1 -transfected cells (N1SD) ( C ), the differentiated stably Notch2- transfected cells (N2SD) ( D ), the differentiated stably Notch3- transfected cells (N3SD) ( E ), the differentiated stably Notch4- transfected cells (N4SD) ( F ), the differentiated stably Dlk1 -transfected cells (DLK1SD) ( H ) and the differentiated stably Dlk2 -transfected cells (DLK2SD) ( I ). ( G ) qPCR analysis of mtCytb (mitochondrial cytochrome b) DNA amplification (related to genomic ApoB DNA amplification; see Materials and Methods section) in seven-day differentiated C3H10T1/2 cells overexpressing Notch ( G ) and Dlk ( J ) genes. Data from qRT-PCR and qPCR assays were normalized to P0 mRNA transcription levels. The fold activation or inhibition levels in PCR assays was calculated relative to the levels of seven-day differentiated empty-vector-transfected cells, which were set arbitrarily at 1 (horizontal black line). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t -test is indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Non-statistical significance is indicated by ns.

Article Snippet: Plasmid pC-N1 (N1S) contains the complete mouse Notch1 gene cDNA (ATCC clone: MBA-105) in sense orientation [ ].

Techniques: Over Expression, Transfection, Staining, Plasmid Preparation, Quantitative RT-PCR, Marker, Expressing, Stable Transfection, Amplification, Activation Assay, Inhibition

Journal: Cells

Article Title: NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

doi: 10.3390/cells9092032

Figure Lengend Snippet: Oxygen consumption rate (OCR) in C3H10T1/2 adipocytes overexpressing Dlk or Notch genes. ( A ) Relative oxygen consumption rate (OCR) in non-differentiated (C3HND) and differentiated (C3HD) C3H10T1/2 cells. Relative oxygen consumption rate (OCR) in differentiated C3H10T1/2 cells overexpressing Dlk ( B ), Notch1 ( C ), Notch2 ( D ), Notch3 ( E ), or Notch4 ( F ) genes. The fold activation or inhibition levels were calculated relative to time 0 of non-differentiated cells, and, additionally, to OCR of non-differentiated cells (ND) ( A ) or seven-day differentiated empty-vector-transfected cells (VD) ( B – F ), 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 -test is indicated at 144 min (* p ≤ 0.05, ** p ≤ 0.01). Non-statistical significance is indicated by ns.

Article Snippet: Plasmid pC-N1 (N1S) contains the complete mouse Notch1 gene cDNA (ATCC clone: MBA-105) in sense orientation [ ].

Techniques: Activation Assay, Inhibition, Plasmid Preparation, Transfection

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 mRNA expression levels. The expression of alpha-tubulin was used as a loading control in all Western blots to normalize expression data. Blot signals from empty vector and over-expressing cells were cropped from original blots and delineated with horizontal white spaces (original blots for each protein signal are shown in Supplementary Figure ). 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: Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation .

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

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: Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation .

Techniques: Over Expression, Microscopy, Quantitative RT-PCR, Expressing, Transfection, 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: 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: Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation .

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: 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: Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation .

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: 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: Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation .

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: Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation .

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: Oligonucleotides used in qRT-PCR assays for the expression of the four Notch genes and the Hey1 gene.

Article Snippet: Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation .

Techniques: Expressing

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: Primary and secondary antibodies used in Western blot analysis.

Article Snippet: Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation .

Techniques: Western Blot