Journal: Oncology Letters

Article Title: miR-29a activates Hes1 by targeting Nfia in esophageal carcinoma cell line TE-1

doi: 10.3892/ol.2014.2678

Figure Lengend Snippet: Overexpression of miR-29a upregulates Hes1 and downregulates Nfia. (A) Notch1, Hes1, and Nfia mRNA expression levels were detected using reverse transcription-quantitative polymerase chain reaction in TE-1 cells transfected with the miR-29a precursor or control virus. * P<0.05. (B) Notch1, Hes1, and Nfia protein expression levels were detected using western blot analysis in TE-1 cells transfected with the miR-29a precursor virus or control virus. (C) TargetScan prediction of the miR-29a binding site within Nfia mRNA. (D) Map of the pmirGLO luciferase reporter vector. The red rectangles indicate the restrictive endonucleases used for cloning. (E) Luciferase activity assay: TE-1 cells were transfected with the miR-29a precursor or scrambled virus for four days, transfected with the reporter vectors for 24 h, and harvested. Protein extracts were prepared and assayed for firefly and Renilla luciferase activity, and firefly luciferase activity was normalized to Renilla luciferase activity. Data are presented as the mean ± standard deviation from three independent experiments. * P<0.05. miR, microRNA; Hes1, hairy and enhancer of split 1; Nfia, nuclear factor 1 A; UTR, untranslated region.

Article Snippet: The membranes were blocked with 5% non-fat milk in PBS and Tween 20 (PBST; Beyotime Institute of Biotechnology) for 1 h at room temperature and incubated with polyclonal rabbit anti-goat Nfia (sc-30918; 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), polyclonal rabbit anti-goat Notch1 (sc-6014; 1:1,000; Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-goat Hes1 (sc-13842; 1:1,000; Santa Cruz Biotechnology, Inc.) or polyclonal rabbit anti-goat β-actin (sc-1616; 1:1,000; Santa Cruz Biotechnology, Inc.), at 4°C overnight.

Techniques: Over Expression, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Control, Virus, Western Blot, Binding Assay, Luciferase, Plasmid Preparation, Cloning, Activity Assay, Standard Deviation

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: Primers and PCR conditions for respective genes

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Amplification

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: Notch1 antisense reduces NOTCH1 expression and NSC self-renewal. NSCs were cultured in the presence of 20 ng/ml EGF in the absence or presence of 20 μmNotch1antisense and harvested after 1 DIV for protein or cultured for a total of 3 DIV (to form P1 neurospheres) and assayed either by single-sphere dissociation (A) or batch culture (B) for the formation of P2 neurospheres.A, Western blot analysis reveals a reduction in NOTCH1-PF1 expression (inset; p < 0.05; t test; n = 3) in antisense-treated P1 neurospheres. A concomitant decrease was observed in the ability of antisense-treated, individual equivalent sized P1 neurospheres to produce P2 neurospheres (*p < 0.05; t test; n = 3) compared with sense treatment. B, Assaying for the ability of P1 neurospheres treated with Notch1 antisense to produce P2 neurospheres by batch culture analysis also reveals a significant decrease in their ability to produce P2 neurospheres (*p < 0.05; t test;n = 3).

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Cell Culture, Western Blot

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: Disruption of NOTCH1 signaling by γ-secretase inhibitor II delays P1 neurosphere formation and reduces their ability to produce P2 neurospheres. A, To ensure that the γ-secretase inhibitor that we were using was effectively blocking production of NOTCH1-PF3, NSCs were cultured in 20 ng/ml EGF (20 ng/ml) for 24 hr, at which point DMSO (carrier) or γ-secretase inhibitor II (50 μm) was added, and the cells were harvested 4 hr later for total proteins and Western blot analysis. A, The asterisk indicates an increase in the P2 proteolytic product of NOTCH1, as would be expected if the γ-secretase inhibitor was effectively blocking production of NOTCH1-PF3 (n = 3), and identifies the upper band as furin-processed NOTCH1 or NOTCH1-PF1. B, Three dayin vitro P1 neurospheres that were treated with γ-secretase inhibitor for 4 hr and harvested for nuclear proteins and Western blot analysis demonstrate a decrease in NOTCH1-PF3 compared with DMSO control. C–H, NSCs were cultured in EGF (20 ng/ml) and either DMSO (C, E,G; carrier) or γ-secretase inhibitor II (D, F, H; 30 μm), and digital micrographs were taken after 6 (C, D), 18 (E,F), and 88 hr (G,H). I, Single-sphere dissociation assay reveals a significant reduction in self-renewal capacity of P1 neurospheres generated for 3 DIV in the presence of γ-secretase inhibitor II (30 μm) compared with DMSO controls (*p< 0.05; t test; n = 3).Inset shows a reduction of NOTCH1-PF1 expression in P1 neurospheres treated for 1 DIV, from the time of plating, with 50 μm γ-secretase inhibitor II compared with the DMSO control (p < 0.05; t test;n = 3), indicating that constitutive inhibition of NOTCH1 activation for at least 24 hr leads to an overall decrease in NOTCH1 expression. Scale bar, 100 μm. N.S., Nonspecific; γ-SI, γ-secretase inhibitor.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Blocking Assay, Cell Culture, Western Blot, Generated, Expressing, Inhibition, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: EGF+CNTF treatment of embryonic P1 neurospheres specifically increases Notch1 mRNA and protein expression. A–D, Immunofluorescence micrographs of a coronal section (8 μm) through the forebrain of an E14 mouse embryo.A, Nuclei were labeled with Hoechst 33258 (blue). CNTFRα-immunoreactive cells in the ventricular zone were visualized with Cy3 (B, red), and Notch1-immunoreactive cells were labeled with FITC (C, green). D, A merged image of B and C, where yellow staining indicates colocalization of NOTCH1 and CNTFRα. Box inA indicates area magnified in B–D.E–G, NSCs were cultured in 20 ng/ml EGF, the absence or presence of 20 ng/ml CNTF, and harvested after 24 hr for total RNA and RT-PCR Southern blot analysis (E,F), or after 3 DIV for Western blot analysis (G). Notch1 expression increased significantly (*p < 0.05 vs EGF; ttest; n = 3) after 1 DIV of CNTF treatment (E) compared with no change inNotch3 expression (F). Both the 93-4 and Santa Cruz intracellular NOTCH1 antibodies reveal an increase in NOTCH1-PF1 and NOTCH1-PF2 proteolytic products after 3 DIV of EGF+CNTF treatment compared with EGF alone (G) (p < 0.01; ttest; n = 5). Nuclear expression of NOTCH1-PF3 increases in 3 DIV P1 neurospheres cultured constitutively in EGF+CNTF compared with EGF alone (H) (p < 0.01; t test;n = 4). Scale bars: A, 50 μm;D, 100 μm. LGE, Lateral ganglionic eminence; LV, lateral ventricle; CTX, cortex.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Immunofluorescence, Labeling, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Southern Blot, Western Blot

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: Cell–cell contact is not required for CNTF to increase NOTCH1 expression but is required for CNTF to increase NSC self-renewal. A, Western blot analysis reveals that NOTCH1-PF1 expression increases as early as 2 hr after CNTF treatment of 3 DIV EGF-derived P1 neurospheres (n = 3).B, Totally dissociated primary neurospheres were cultured in EGF+CNTF for 6 hr; Western blot analysis demonstrates a threefold increase in NOTCH1-PF1 expression (p < 0.05; t test;n = 3) compared with EGF controls. No increase in NOTCH1-PF2 could be detected. C, P1 neurospheres were generated in EGF or in EGF+CNTF in the absence (0–24 hr) or presence (72–96 hr) of cell–cell contact. After 7 DIV the three different groups were assayed for the formation of P2 neurospheres by single-sphere dissociation and culture in EGF alone (each group was washed at 24 and 96 hr). Compared with EGF, addition of CNTF for 24 hr at 3 DIV increased the formation of P2 neurospheres by 59% (p < 0.0001; Tukey HSD test;n = 3), whereas there was no difference in P2 neurosphere formation when CNTF was added for the first 24 hr (p > 0.58; Tukey HSD test;n = 3).

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Western Blot, Derivative Assay, Cell Culture, Generated

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: CNTF increases the number of intensely NOTCH1-immunoreactive cells. Primary neurospheres were dissociated and then cultured on poly-l-ornithine-coated coverslips for 6 hr in either EGF (A, B) or EGF+CNTF (C, D), and blind counts were made (as described in Materials and Methods) on the number of intensely NOTCH1-immunoreactive cells (C).A, C, Nuclei were labeled using Hoechst 33258 (blue). NOTCH1-immunoreactive cells were labeled with rhodamine (B, D,red). E, Compared with cells cultured in EGF alone, cells cultured in EGF+CNTF demonstrate a 49% increase in the number of intensely NOTCH1-immunoreactive cells (*p < 0.003; t test;n = 3). Arrowheads indicate examples of cells that stain intensely for NOTCH1, and small arrows indicate examples of cells that stain weakly for NOTCH1. Scale bar, 20 μm.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Cell Culture, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: IL6+sIL6R increases NOTCH1 expression and P2 neurosphere production in P1 neurospheres generated fromLIFRβ−/−mice. P1 neurospheres were generated from wild-type (+/+) or null mutant (−/−) LIFRβ littermates, in the various conditions indicated, and were then assayed after 3 DIV for NOTCH1 protein with Western blot and after 7 DIV for P2 neurosphere production by single-sphere dissociation in EGF alone. Increase in P2 neurosphere production in wild-type (+/+) EGF+CNTF generated P1 neurospheres and inLIFRβ−/−P1 neurospheres generated in EGF+IL6+sIl6R correlated with concomitant increases in NOTCH1-PF1 expression (inset). CNTF had no effect on P2 neurosphere production or NOTCH1-PF1 expression inLIFRβ−/−P1 neurospheres. **p < 0.01 versus +/+ control culture or −/− control culture; Tukey HSD test; n= 5. N-PF1, NOTCH1-PF1.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Generated, Mutagenesis, Western Blot

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: The CNTF-induced increase in NOTCH1 expression in dissociated primary neurospheres is dependent on either EGF or FGF2 signaling. A, B, Single-cell suspensions derived from primary neurospheres and cultured for 6 hr in the indicated conditions reveal that CNTF had no effect on NOTCH1-PF1 expression in the absence of EGF (B) (n = 3) and that CNTF can increase NOTCH1-PF1 expression in either EGF- or FGF2-containing media (B) (p< 0.05; t test; n = 3).

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Derivative Assay, Cell Culture

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: CNTF enhances the expression of NOTCH1 in vivo. Adult CD1 mice were infused with either EGF (A, C, D) or EGF+CNTF (B, E, F) for 6 d, after which brains were processed for NOTCH1 immunohistochemistry. Infusion of EGF+CNTF resulted in an overall increase in NOTCH1 staining intensity as well as a markedly thickened layer of NOTCH1 expression on the lateral aspect (A, B, arrows) of the ventricle. Furthermore, more cells in the ventricular zone labeled for NOTCH1 in EGF+CNTF (C, NOTCH1, 76 ± 12%;D, Hoechst) compared with EGF (E, NOTCH1, 39 ± 7%; F, Hoechst) infused mice (p < 0.026; t test;n = 3 each group). C–F,Arrows indicate NOTCH1 unlabeled cells. Scale bars:B, 100 μm; F, 25 μm.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, In Vivo, Immunohistochemistry, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: CNTF treatment changes the expression of genes regulated by NOTCH1 signaling. A–C, Primary neurospheres derived from the E14 striatum were grown in the presence of EGF for 7 DIV, dissociated, and then cultured (5 × 104 cells/ml) in either EGF or EGF+CNTF. The cells were then harvested for total RNA or protein at 3 DIV and processed for RT-PCR Southern or Western blot analyses as described in Materials and Methods. A, Constitutive CNTF treatment significantly decreases Hes5 expression, whereas Hes1expression in CNTF-treated P1 neurospheres does not differ significantly from 3 DIV EGF-derived P1 neurospheres. B,Mash1 expression, mRNA, and protein are significantly reduced in P1 neurospheres cultured for 3 DIV in the presence of EGF+CNTF compared with EGF alone. C,Delta3 expression is reduced in 3 DIV EGF+CNTF P1 neurosphere cultures compared with EGF cultures. *p< 0.05 versus EGF; t test (A–C;n = 3).

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Derivative Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot