Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T-cells (NFAT)c2 Inhibits Notch Receptor Signaling in Osteoblasts

doi: 10.1074/jbc.m112.340455

Figure Lengend Snippet: FIGURE 1. NICD induces NFATc2 expression in osteoblasts by post-tran- scriptional mechanisms. RosaNotch calvarial osteoblasts were infected with Ad-CMV-CRE (CRE, black bars or full circles) or control Ad-CMV-GFP (GFP, white bars or open circles). Wild-type C57BL/6 osteoblasts were exposed to immo- bilized Dll1 (Dll1, black bars), or control BSA (BSA, white bars). In A and C, RosaNotchosteoblastswereculturedfor3days(A)or1day(C)afterconfluence, and total RNA was extracted, reverse-transcribed, and amplified by qRT-PCR. Data are expressed as ratio of Nfatc1, Nfatc2, Nfatc3, Nfatc4 (A), and NFATc2 hnRNA (C) copy number corrected for Rpl38 expression, relative to corrected expression in GFP. Values are means S.E., n 4. *, significantly different between CRE and GFP, p 0.05. Data are from representative experiments performed in duplicate. In B, total cellular extracts from RosaNotch osteoblasts were fractioned by gel electrophoresis and transferred to an Immobilon-P membrane, which was incubated with an antibody against NFATc2, stripped, and reprobed with an antibody against actin. Digital images were acquired, and signal from NFATc2 and actin antibodies was quantified by densitometry to estimate protein expression. Data are expressed as ratio of NFATc2 levels corrected for actin expression relative to corrected NFATc2 levels in GFP. Val- ues are means of four independent experiments S.E. *, significantly differ- ent between CRE and GFP, p 0.05. In D, osteoblasts from wild-type C57BL/6 were cultured for 3 days after confluence, and total RNA was extracted, reverse-transcribed, and amplified by qRT-PCR. Data are expressed as ratio of Hey2 or Nfatc2 copy number corrected for Rpl38 expression, relative to cor- rected expression in BSA. Values are means S.E., n 4. *, significantly dif- ferent between Dll1 and BSA, p 0.05. In E, RosaNotch osteoblasts were tran- scriptionally arrested with DRB (time 0) and harvested at the indicated times. Total RNA was extracted and amplified by qRT-PCR, and data are expressed as the percentage of Nfatc2 mRNA copy number corrected for Rpl38 expression, relative to the corrected Nfatc2 mRNA expression before DRB treatment, plot- ted versus time. Data are means S.E., n 4. Nfatc2 decay slopes for CRE and GFP are statistically different, p 0.05. Data are from representative experi- ments performed in triplicate.

Article Snippet: An adenoviral vector expressing green fluorescent protein (GFP) under the control of the CMV promoter (Ad-CMV-GFP, Vector Biolabs) was used as control.

Techniques: Expressing, Infection, Control, Reverse Transcription, Amplification, Quantitative RT-PCR, Nucleic Acid Electrophoresis, Membrane, Incubation, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T-cells (NFAT)c2 Inhibits Notch Receptor Signaling in Osteoblasts

doi: 10.1074/jbc.m112.340455

Figure Lengend Snippet: FIGURE 2. NFATc2 is expressed and activated in osteoblasts. RosaNotch or wild-type C57BL/6 calvarial osteoblasts were transduced with Ad-CMV-caN- FATc2 tagged with HA (NFATc2, black bars) or control Ad-CMV-GFP (Control, whitebars).RosaNotchosteoblastswereco-transducedwithAd-CMV-CRE(CRE) to induce Notch, or control Ad-CMV-GFP (GFP), and cultured for 72 h. In A, total cellular extracts from RosaNotch osteoblasts were fractioned by gel elec- trophoresis and transferred to an Immobilon-P membrane, which was incu- bated with an antibody against NFATc2. Membranes were stripped and rep- robedwithanantibodyagainstactin.InBandD,totalRNAwasextractedfrom RosaNotchosteoblastsandamplifiedbyqRT-PCR.Dataareexpressedasratioof Nfatc2 and Rcan1.4 copy number corrected for Rpl38 expression, relative to corrected expression in control cells co-transduced with Ad-CMV-GFP. Values are means S.E., n 4. *, significantly different between NFATc2 and control, p 0.05. , significantly different between CRE and GFP, p 0.05. In C, wild-type C57BL/6 osteoblasts were cultured to confluence, and DNA was cross-linked to associated proteins and fragmented by sonication. DNA-pro- tein complexes were obtained by immunoprecipitation with an HA antibody or control IgG, and DNA was purified following reversal of the cross-linking reaction. Immunoprecipitated DNA and fragmented input DNA were ampli- fied by qPCR with primers flanking Nfatc consensus sequences required for the activity of the Rcan1.4 promoter. Data are expressed as enrichment of DNA by immunoprecipitation with the HA antibody relative to control IgG, corrected for input DNA levels. Values are means of three qPCR reactions. Data are from representative experiments performed in duplicate.

Article Snippet: An adenoviral vector expressing green fluorescent protein (GFP) under the control of the CMV promoter (Ad-CMV-GFP, Vector Biolabs) was used as control.

Techniques: Transduction, Control, Cell Culture, Membrane, Expressing, Sonication, Immunoprecipitation, Purification, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T-cells (NFAT)c2 Inhibits Notch Receptor Signaling in Osteoblasts

doi: 10.1074/jbc.m112.340455

Figure Lengend Snippet: FIGURE 3. NFATc2 suppresses Notch canonical signaling in osteoblasts. RosaNotch calvarial osteoblasts were transduced with Ad-CMV-caNFATc2 (NFATc2, black bars) or control Ad-CMV-GFP (Control, white bars) and co-trans- duced with Ad-CMV-CRE (CRE) to induce Notch or control Ad-CMV-GFP (GFP). In A, osteoblasts were cultured to subconfluence and transiently transfected with the 12CSL-Luc reporter or with the Hey1-Luc or Hey2-Luc promoter constructs, co-transfected with a CMV/-galactosidase expression vector, and harvested after 48 h. Data shown represent luciferase/-galactosidase activity. Values are means S.E., n 6. In B, osteoblasts were cultured for 72 h after confluence, and total RNA was extracted and amplified by qRT-PCR. Data are expressed as ratio of Hey1 or Hey2 copy number, corrected for Rpl38 expression, relative to corrected expression in control cells co-transduced with Ad-CMV-GFP. Values are means S.E., n 4. *, significantly different betweenNFATc2andcontrol,p0.05.,significantlydifferentbetweenCRE and GFP, p 0.05. Data are from representative experiments performed in duplicate.

Article Snippet: An adenoviral vector expressing green fluorescent protein (GFP) under the control of the CMV promoter (Ad-CMV-GFP, Vector Biolabs) was used as control.

Techniques: Transduction, Control, Cell Culture, Transfection, Construct, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Amplification, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T-cells (NFAT)c2 Inhibits Notch Receptor Signaling in Osteoblasts

doi: 10.1074/jbc.m112.340455

Figure Lengend Snippet: FIGURE 4. NFATc2 and CSL bind to similar DNA consensus sequences in osteoblasts. Binding of nuclear proteins to DNA was tested by electropho- retic mobility shift assay in RosaNotch calvarial osteoblasts transduced with Ad-CMV-caNFATc2 (NFATc2) or control Ad-CMV-GFP (Control) and co-trans- duced with Ad-CMV-CRE (CRE) to induce Notch or control Ad-CMV-GFP (GFP). Cells were cultured to confluence, and nuclear extracts were incubated with [-32P]ATP-labeled oligonucleotides. In A, a radiolabeled oligonucleotide containing a Csl consensus sequence from the EBNA2 promoter was used. Competition of binding reactions was performed in the presence of unla- beled Csl consensus and mutant oligonucleotides or unlabeled Nfatc oligo- nucleotides containing homologous or mutated consensus sequences from the Bnp promoter in 200-fold excess. In B, a radiolabeled oligonucleotide con- taining an Nfatc consensus sequence from the Bnp promoter was used. Com- petition of binding reactions was performed in the presence of unlabeled Nfatc consensus and mutant oligonucleotide or unlabeled Csl oligonucleo- tides containing homologous or mutated consensus sequences from the EBNA2 promoter in 200-fold excess. DNA-nuclear protein complexes were resolved by gel electrophoresis and visualized by autoradiography, and arrows indicate the position of complexes. Autoradiographs are representa- tive of four independent experiments.

Article Snippet: An adenoviral vector expressing green fluorescent protein (GFP) under the control of the CMV promoter (Ad-CMV-GFP, Vector Biolabs) was used as control.

Techniques: Binding Assay, Mobility Shift, Transduction, Control, Cell Culture, Incubation, Labeling, Sequencing, Mutagenesis, Nucleic Acid Electrophoresis, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T-cells (NFAT)c2 Inhibits Notch Receptor Signaling in Osteoblasts

doi: 10.1074/jbc.m112.340455

Figure Lengend Snippet: FIGURE 6. NICD and NFATc2 regulate osteoblast function. RosaNotch calvar- ial osteoblasts were transduced with Ad-CMV-caNFATc2 (NFATc2, black bars) orcontrolAd-CMV-GFP(Control,whitebars)andco-transducedwithAd-CMV- CRE (CRE) to induce Notch or control Ad-CMV-GFP (GFP), and cells were cul- tured for 7–14 days in conditions favoring osteoblast differentiation. In A, following 7 days of culture, total RNA was extracted and amplified by qRT- PCR. Data are expressed as ratio of Dlx5, Runx2, Alpl, Bsp, osteocalcin, Col1a1, and osteopontin copy number, corrected for Rpl38 expression, relative to corrected expression in control cells co-transduced with Ad-CMV-GFP. Values are means S.E., n 4, and representative results from two independent experiments are shown. In B, cells were cultured for 7 days and extracted with Triton X-100 for determination of alkaline phosphatase activity, expressed as nanomoles of p-nitrophenol/min/g of total protein. Values are means from fourindependentexperimentsS.E.InC,cellsculturedfor14dayswerefixed and stained with Alizarin Red, digital images were acquired, and the mineral- ized area was quantified with the measure function of ImageJ software. A representative culture from two independent experiments is shown. Values are means S.E., n 6. *, significantly different between NFATc2 and control. , significantly different between CRE and GFP, p 0.05. In A and B, selected data from control cells co-transduced with CRE or GFP were published previ- ously (14).

Article Snippet: An adenoviral vector expressing green fluorescent protein (GFP) under the control of the CMV promoter (Ad-CMV-GFP, Vector Biolabs) was used as control.

Techniques: Transduction, Control, Amplification, Quantitative RT-PCR, Expressing, Cell Culture, Activity Assay, Staining, Software

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor of Activated T-cells (NFAT)c2 Inhibits Notch Receptor Signaling in Osteoblasts

doi: 10.1074/jbc.m112.340455

Figure Lengend Snippet: FIGURE5.NFATc2opposestheassociationofCSLtotheHey2promoterin osteoblasts. Binding of NFATc2 and CSL to the Hey2 promoter was tested by ChIP in RosaNotch calvarial osteoblasts transduced with Ad-CMV-caNFATc2 tagged with HA (NFATc2) or control Ad-CMV-GFP (Control) and co-transduced with Ad-CMV-CRE (CRE) to induce Notch or control Ad-CMV-GFP (GFP). Cells were cultured to confluence, DNA cross-linked to associated proteins, and fragmented by sonication. DNA-protein complexes were obtained by immu- noprecipitation (I.P.) with an HA (A) or a CSL (B) antibody or control IgG, and DNA was purified following reversal of the cross-linking reaction. Immuno- precipitated DNA and fragmented input DNA were amplified by qPCR with primers flanking Csl consensus sequences that regulate the activity of the Hey2 promoter. Data are expressed as enrichment of DNA by immunoprecipi- tation with the HA or CSL antibody relative to control IgG, corrected for input DNA levels. Values are means of three qPCR reactions.

Article Snippet: An adenoviral vector expressing green fluorescent protein (GFP) under the control of the CMV promoter (Ad-CMV-GFP, Vector Biolabs) was used as control.

Techniques: Binding Assay, Transduction, Control, Cell Culture, Sonication, Purification, Amplification, Activity Assay