Journal: Molecular medicine reports

Article Title: Disorder of the mevalonate pathway inhibits calcium-induced differentiation of keratinocytes.

doi: 10.3892/mmr.2017.7128

Figure Lengend Snippet: Figure 2. Treatment of KCs with inhibitors of the mevalonate pathway reduced the expression levels of Notch1 and p53. Representative western blot images and quantification of protein expression levels of Notch1 and p53 in KCs treated with PRA, ALD, FTI‑277 and GGTI‑298 inhibitors (A) alone, (B) in combination with two inhibitors or (C) in combination with three or four inhibitors for 48 h, in the presence or absence of Ca2+. Data are expressed as mean ± standard error of three independent experiments. *P<0.01 and #P<0.05 vs. untreated control in the presence of Ca2+. PRA, pravastatin; ALD, alendronate; FTI‑277, farnesyl transferase inhibitor; GGTI‑298, geranylgeranyl transferase inhibitor; KCs, keratinocytes; NEXT, Notch1 extracellular truncation; NCID, Notch1 intracellular domain.

Article Snippet: Anti-K1(mouse monoclonal IgG2a; sc-376224), involucrin (INV; rabbit polyclonal IgG; sc-28557), Notch1 (goat polyclonal IgG; sc-6014), p53 (rabbit polyclonal IgG; sc-6243), phosphorylated (p)-extracellular signal-regulated kinase (ERK; rabbit polyclonal IgG; sc-101761), p-protein kinase B (AKT; rabbit polyclonal IgG; sc-33437) and β-actin (mouse monoclonal IgG1; sc-376421) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

doi: 10.1371/journal.pone.0147846

Figure Lengend Snippet: After C57BL/6 mice were housed in a CEC with scopolamine administration for 10 days, LGs were obtained and prepared for qRT-PCR, and (A) mRNA levels of NOTCHs, DLLs, and JAGs were measured. (B) The change in the mRNA level of NOTCH1/DLL4 during DE induction was measured (C) During DE induction, each LG samples were prepared for immunoblot for NOTCH1 and DLL4 at Day 2, Day 4, Day 6, Day 8, and Day 10. Densitometry for protein concentreation quantification was done by using ImageJ software. Student’s t-test for statistical analysis: *p<0.05, **p<0.01, †p<0.001. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye).

Article Snippet: Sections were blocked with rabbit/goat/rat serum for 40 minutes at room temperature and exposed to primary antibodies: NOTCH1 (Goat polyclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc., Dallas, TX), DLL4 (Rabbit polyclonal anti-mouse, 1μg/ml; Abcam ® , Inc.), LYVE-1 (Rat monoclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc.).

Techniques: Quantitative RT-PCR, Western Blot, Software, Standard Deviation, Control

Journal: PLoS ONE

Article Title: Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

doi: 10.1371/journal.pone.0147846

Figure Lengend Snippet: While C57BL/6 mice were housed in a CEC with scopolamine administration for 10 days, several groups of mice were administered anti-Dll4 Ab and GSI for inhibiting the NOTCH1-DLL4 axis. Anti-IgG Ab was administered as a control for the anti-Dll4 antibody group, and DMSO was administered as a negative control. LGs were obtained after 10 days of DE induction and were prepared for qRT-PCR, immunoblot, and immunostaining. (A) The mRNA levels of LYVE-1, VEGF-C, VEGF-D, and VEGFR3 were measured using qPCR for each group. (B) Immunoblot and densitometry of LYVE-1 were measured for each group. (C) Immunofluorescence staining of LYVE-1 was performed for each group. Student’s t-test for statistical analysis: *p<0.05, **p<0.01. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye; α-Dll4 = anti-Dll4 antibody; α-IgG = anti-IgG antibody; GSI = γ-secretase inhibitor; DMSO = dissolved dimethyl sulfoxide).

Article Snippet: Sections were blocked with rabbit/goat/rat serum for 40 minutes at room temperature and exposed to primary antibodies: NOTCH1 (Goat polyclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc., Dallas, TX), DLL4 (Rabbit polyclonal anti-mouse, 1μg/ml; Abcam ® , Inc.), LYVE-1 (Rat monoclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc.).

Techniques: Control, Negative Control, Quantitative RT-PCR, Western Blot, Immunostaining, Immunofluorescence, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

doi: 10.1371/journal.pone.0147846

Figure Lengend Snippet: WT B6 and HIF-1α CKO mice were housed in a CEC with scopolamine administration for 10 days. LGs were obtained and prepared for qRT-PCR and (A) The mRNA level of NOTCH1, DLL4, LYVE-1, and Podoplanin at Day 10 was measured. (B) Immunoblot and densitometry for NOTCH1 and LYVE-1 were measured at Day 10. (C) Immunofluorescence staining of LYVE-1 was performed for DE-induced WT B6 mice and DE-induced HIF-1α CKO mice at Day 10. Student’s t-test for statistical analysis: *p<0.05, **p<0.01. Error bars indicate standard deviation. (WT = wild-type; DE = dry eye; HIF-1α CKO = HIF-1α conditional knockout).

Article Snippet: Sections were blocked with rabbit/goat/rat serum for 40 minutes at room temperature and exposed to primary antibodies: NOTCH1 (Goat polyclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc., Dallas, TX), DLL4 (Rabbit polyclonal anti-mouse, 1μg/ml; Abcam ® , Inc.), LYVE-1 (Rat monoclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc.).

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Standard Deviation, Knock-Out

Journal: PLoS ONE

Article Title: Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

doi: 10.1371/journal.pone.0147846

Figure Lengend Snippet: During 10 days of DE induction, (A) LGs were obtained on Days 0, 2, 4, 6, and 10 for IHC staining of CD45 + cells. (B) The fold change of CD45 + cells was measured at Days 0, 2, 4, 6, and 10. (C) During DE induction, several groups of mice were administered anti-Dll4 Ab and GSI to inhibit the NOTCH1-DLL4 axis. Anti-IgG Ab was administered as a control for the anti-Dll4 Ab group, and DMSO was administered as a negative control. LGs were obtained at Day 10 of DE induction and were prepared for IHC staining of CD45 + cells. (D) The actual percentage of CD45 + cells was calculated. (E) Flow cytometry for CD45 + cells was performed for each condition according to the manufacturer’s protocol. (F) DE was induced for 10 days in B6 and HIF-1CKOmice. LGs were obtained and prepared for IHC staining. (G) The actual percentage of CD45 + cells was calculated for non-DE HIF-1α CKO mice and the DE HIF-1α CKO mice. (H) Flow cytometry was performed for the two groups. Student’s t-test for statistical analysis: *p<0.05, **p<0.01, †p<0.001. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye; α-Dll4 = anti-Dll4 antibody; α-IgG = anti-IgG antibody; GSI = γ-secretase inhibitor; DMSO = dissolved dimethyl sulfoxide; HIF-1α CKO = HIF-1α conditional knockout).

Article Snippet: Sections were blocked with rabbit/goat/rat serum for 40 minutes at room temperature and exposed to primary antibodies: NOTCH1 (Goat polyclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc., Dallas, TX), DLL4 (Rabbit polyclonal anti-mouse, 1μg/ml; Abcam ® , Inc.), LYVE-1 (Rat monoclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc.).

Techniques: Immunohistochemistry, Control, Negative Control, Flow Cytometry, Standard Deviation, Knock-Out

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: Antibody Details

Article Snippet: Notch1 , Rabbit , 1:250 – 1:50 , Cell Signaling , D1E11 , residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region.

Techniques: Concentration Assay, Purification, Marker, Sequencing

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ reduces surface expression of Notch components. HUVECs were transfected with siRHOQ and the effects on DLL4 or Notch1 expression were assessed 24 h later by a staining cells for surface protein surface expression, analysed by FACs and expressed relative to control, with example representative image provided, b membrane fraction by western blotting, using CD31 as a loading control or c by QPCR, expressed relative to GAPDH (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test between data group and control; data representative of n = 3 independent experiments)

Article Snippet: Membrane were probed with primary antibodies used were RHOQ (T8950, Rabbit; Sigma-Aldrich), and Notch1 (3608, Rabbit), Cleaved Notch1 NICD (2421, Rabbit), DLL4 (2421, Rabbit), VEGFR2 (2479, Rabbit), phosphVEGFR2 (2478, Rabbit), B-Actin (3700, Mouse) from Cell signalling and Exocyst70 (sc-365825, Mouse), Cleaved Notch1 (sc-23307, Goat) from Santa-Cruz.

Techniques: Expressing, Transfection, Staining, Control, Membrane, Western Blot

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ increases autophagy and leads to degradation of Notch1 receptor. HUVECs cultured on BSA or rhDLL4 (1 μg/ml)-coated plates and effects on autophagosomes were assessed 16 h later by staining cells with autophagy tracker (red) with changes in tracker levels, a visualised by confocal microscopy (nuclei stained with DAPI (blue)), and assessed by b FACs or c cells were fixed and immuno-fluorescence staining for localisation changes in RHOQ (red) or Notch1 (red) and LC3B (green) proteins and visualised by confocal microscopy (nuclei stained with DAPI (blue)) or d illustrating LC3B binding sites on Notch1 (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test between data group and control, Scale bar = 20 nm; representative images and data of n = 3 independent experiments)

Article Snippet: Membrane were probed with primary antibodies used were RHOQ (T8950, Rabbit; Sigma-Aldrich), and Notch1 (3608, Rabbit), Cleaved Notch1 NICD (2421, Rabbit), DLL4 (2421, Rabbit), VEGFR2 (2479, Rabbit), phosphVEGFR2 (2478, Rabbit), B-Actin (3700, Mouse) from Cell signalling and Exocyst70 (sc-365825, Mouse), Cleaved Notch1 (sc-23307, Goat) from Santa-Cruz.

Techniques: Cell Culture, Staining, Confocal Microscopy, Fluorescence, Binding Assay, Control

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Loss of RHOQ expression leads to NICD degradation in lysosomes. HUVECs cultured on BSA or rhDLL4 (1 μg/ml)-coated plates and effects on lysosomes were assessed 16 h later by staining cells with lysosome tracker (red) with changes in tracker levels, a visualised by confocal microscopy (nuclei stained with DAPI (blue)) and assessed by b FACs or cells were fixed and immuno-fluorescence staining for localisation changes in proteins c duo-link staining detecting only Lamp/Notch1 (red) or Lamp1/NICD (green) associated antibodies, visualised by confocal microscopy. Nuclei stained with DAPI (blue). d Transfected HUVECs with RHOQ siRNA duplexes were cultured on BSA or rhDLL4 (1 μg/ml)-coated plates for 8 h with or without chloroquine (10 µM) before ( a ) changes in DLL4/Notch downstream target expression by QPCR (Error bars = S.D. Key: * p < 0.05, ** p < 0.01, *** p < 0.0001 one-way ANOVA or unpaired Student’s t -test between data group and control, Scale bar = 20 nm; representative images and data of n = 3 independent experiments)

Article Snippet: Membrane were probed with primary antibodies used were RHOQ (T8950, Rabbit; Sigma-Aldrich), and Notch1 (3608, Rabbit), Cleaved Notch1 NICD (2421, Rabbit), DLL4 (2421, Rabbit), VEGFR2 (2479, Rabbit), phosphVEGFR2 (2478, Rabbit), B-Actin (3700, Mouse) from Cell signalling and Exocyst70 (sc-365825, Mouse), Cleaved Notch1 (sc-23307, Goat) from Santa-Cruz.

Techniques: Expressing, Cell Culture, Staining, Confocal Microscopy, Fluorescence, Transfection, Control

Journal: Angiogenesis

Article Title: RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain

doi: 10.1007/s10456-020-09726-w

Figure Lengend Snippet: Summary: RHOQ regulates Notch signalling by transporting cleaved Notch1 to the nucleus, otherwise Notch1 is degraded. a Binding of Notch1 with DLL4 leads to cleavage of Notch1 by ADAM/TACE complex (generating the Notch extracellular truncation (NEXT). Membrane bound gamma-secretase leads to the release of the NICD. The NICD combines with the RHOQ/Exo70 positive nucleus bound endosome, which transports the NICD to the nucleus and subsequent modulation of downstream targets of DLL4/Notch signalling. b In RHOQ negative cells NICD accumulate within the cytoplasm. Loss of translocation of the NICD to the nucleus prevents the induction of the downstream targets of Notch signalling within the nucleus. Rather the NICD is targeted for degradation in lysosomes

Article Snippet: Membrane were probed with primary antibodies used were RHOQ (T8950, Rabbit; Sigma-Aldrich), and Notch1 (3608, Rabbit), Cleaved Notch1 NICD (2421, Rabbit), DLL4 (2421, Rabbit), VEGFR2 (2479, Rabbit), phosphVEGFR2 (2478, Rabbit), B-Actin (3700, Mouse) from Cell signalling and Exocyst70 (sc-365825, Mouse), Cleaved Notch1 (sc-23307, Goat) from Santa-Cruz.

Techniques: Binding Assay, Membrane, Translocation 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: 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