Journal: Molecular Cancer

Article Title: Cooperation of Notch and Ras/MAPK signaling pathways in human breast carcinogenesis

doi: 10.1186/1476-4598-8-128

Figure Lengend Snippet: Overexpression of Notch receptors and ligands in breast cancer . Photomicrographs represent staining of normal and cancerous breast tissue sections with antibodies that recognize (A) Notch1, Notch2, Notch3, Notch4, and (B) Jagged1, Jagged2, Delta-like 1 and Delta-like 4. Inset and arrow in (A) shows nuclear localization of Notch3 in breast cancers. Sections stained in the absence of primary antibodies served as negative controls (-1 0 ). Samples were counterstained with haemotoxylin, and images taken at a magnification of 20×. (C) Scatter plot represents expression of Notch receptors and ligands across various normal (blue) and cancer (red) breast tissue samples analyzed. The total number of cases analyzed under each category (n) is mentioned below each column, and the black bar represents the median score.

Article Snippet: After blocking the nonspecific binding with 4% non fat dry milk, the sections were incubated overnight with the respective primary antibodies at 4°C: Notch1, Notch2, Notch3, Notch4, Delta-like1, Delta-like4, Jagged1, Jagged2, Hes5, Hes1, Numb, cleaved Notch1 antibodies (Santa Cruz Biotechnology, Inc, CA.) and pErk1&2 (Cell Signaling Technology, Inc, CA).

Techniques: Over Expression, Staining, Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells

doi: 10.1038/jcbfm.2012.123

Figure Lengend Snippet: Subcellular localization of NOTCH3 in CADASIL vascular smooth muscle cells (VSMCs). For detection of cell surface NOTCH3, VSMCs were fixed with 3.5% phosphate-buffered paraformaldehyde. NOTCH3 forms more aggregates on the cell surface in CADASIL than in control VSMCs ( A – I ). For intracellular NOTCH3 detection, VSMCs were fixed with methanol. Permeabilized VSMCs show that NOTCH3 does not aggregate inside the cell in either CADASIL or control VSMCs ( J – L ). Immunostainings were performed with antibody (1E4) directed against N3ECD recognizing full-length NOTCH3 and N3ECD. Number of cell lines analyzed: HUmbVSMC: CADASIL n =4, control n =4, HPlaVSMC CADASIL n =4, control n =4, HArtVSMC: CADASIL n =3, HCerVSMC: CADASIL n =2, CADASIL-hmz n =1, control n =3.

Article Snippet: Primary antibodies used in Western blot and immunocytochemistry were NOTCH3 clone 1E4, NOTCH3 clone 5E1, both directed against N3ECD (both kind gifts from Dr A Joutel), NOTCH3 ICD (D11B8, Cell Signaling, Beverly, MA, USA), α -SMA (Sigma), HES5 (M-104, Santa Cruz Biotechnology, Heidelberg, Germany) β -Actin (Sigma), vinculin (Biohit, Helsinki, Finland), integrin β 1, tensin (BD Transduction Laboratories, Mississauga, Canada).

Techniques: Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells

doi: 10.1038/jcbfm.2012.123

Figure Lengend Snippet: Total NOTCH3 and α -SMA expression in CADASIL and control vascular smooth muscle cells (VSMCs). ( A ) Western blot analysis of the expression of NOTCH3 detected with 5E1 directed against N3ECD in CADASIL and control VSMCs. In all, 40 μ g of cell lysate was loaded on each lane. The 280-kDa full-length NOTCH3 (N3FL) and 220 kDa N3ECD are indicated with arrowheads. The 245-kDa band in between (white arrowhead) is unknown. ( B ) The expression of NOTCH3 is significantly higher in HCerVSMCs and HArtVSMCs than in HUmbVSMCs or HPlaVSMCs. There are no statistically significant differences in the total expression level of NOTCH3 or N3FL/N3ECD ratio between CADASIL versus the corresponding control or between control versus heterozygous or homozygous VSMCs. ( C ) Western blot analysis of α -SMA expression in CADASIL and control VSMCs. In all, 20 μ g of cell lysate was loaded on each lane. ( D ) Even though, CADASIL cells have altered actin filament organization, the total amount of α -SMA in CADASIL VSMCs is not significantly different from that in corresponding control VSMCs. The bar graphs show the average intensities and ±s.e.m. from two independent experiments. Both experiments were repeated two times with cells at passage 5.

Article Snippet: Primary antibodies used in Western blot and immunocytochemistry were NOTCH3 clone 1E4, NOTCH3 clone 5E1, both directed against N3ECD (both kind gifts from Dr A Joutel), NOTCH3 ICD (D11B8, Cell Signaling, Beverly, MA, USA), α -SMA (Sigma), HES5 (M-104, Santa Cruz Biotechnology, Heidelberg, Germany) β -Actin (Sigma), vinculin (Biohit, Helsinki, Finland), integrin β 1, tensin (BD Transduction Laboratories, Mississauga, Canada).

Techniques: Expressing, Control, Western Blot

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells

doi: 10.1038/jcbfm.2012.123

Figure Lengend Snippet: Actin cytoskeleton in CADASIL vascular smooth muscle cells (VSMCs). VSMCs fixed with methanol were immunostained for smooth muscle cell α -actin ( α -SMA). α -SMA filament have altered organization in CADASIL VSMCs. In all CADASIL VSMCs the α -SMA positive actin filaments are short, poorly organized and form nodes (clusters of short haphazardly oriented filaments, selected five marked with arrowheads) ( A : HUmbVSMC, B : HCerVSMC, C : HCerVSMC-G528C, F : HCerVSMC-hmz, and G : HArtVSMC). Actin filament nodes in CADASIL VSMCs are shown in higher magnification in an inset ( I : HCerVSMC-hmz). Control VSMCs have robust cell spanning actin filaments ( D : HUmbVSMC and E : HCerVSMC and an inset H : HCerVSMC). Number of cell lines analyzed: HUmbVSMC: CADASIL n =4, control n =4, HPlaVSMC CADASIL n =4, control n =4, HArtVSMC: CADASIL n =3, HCerVSMC: CADASIL n =2, CADASIL-hmz n =1, control n =3. Control HUmbVSMCs were transduced with nontarget short hairpin RNA (shRNA) ( J ) or NOTCH3 -targeted shRNA2 ( K ). Silencing of NOTCH3 expression induced similar alterations to organization of actin filaments as observed in CADASIL VSMCs. Silencing of NOTCH3 expression was verified by Western blot ( L ). NOTCH3-targeted shRNA1 and shRNA2 reduced relative intensity of NOTCH3 expression by 36 and 63% when compared with nontarget shRNA. NOTCH3 signaling activity is also reduced as indicated by lower expression level of NOTCH3 target gene HES5 (shRNA1 5% and shRNA2 30%). Relative intensity is calculated from an average of combined N3FL and N3ICD intensities. Intensities are averages from two independent experiments normalized to β -actin. Average intensities are shown in proportion to nontarget shRNA sample (relative intensity). N3FL and N3ICD were detected by antibody directed against N3ICD.

Article Snippet: Primary antibodies used in Western blot and immunocytochemistry were NOTCH3 clone 1E4, NOTCH3 clone 5E1, both directed against N3ECD (both kind gifts from Dr A Joutel), NOTCH3 ICD (D11B8, Cell Signaling, Beverly, MA, USA), α -SMA (Sigma), HES5 (M-104, Santa Cruz Biotechnology, Heidelberg, Germany) β -Actin (Sigma), vinculin (Biohit, Helsinki, Finland), integrin β 1, tensin (BD Transduction Laboratories, Mississauga, Canada).

Techniques: Control, Transduction, shRNA, Expressing, Western Blot, Activity Assay

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Time- and dose-dependent inhibition of NAC on the intracellular domain of Notch3 (N3IC), but not Notch1 (N1IC). HeLa cells were treated with NAC (2-10 mM) for 0-24 h. B. Dose-dependent inhibition by NAC (0-10 mM, 6 h) on the protein expression of N3IC in HeLa cells. C. NAC treatment (5 mM, 0-12 h) reduces protein levels of N3IC and extracellular domain of Notch 3 (N3EC) but not full length Notch 3 precursor (N3FL) in HeLa cells. Densitometry quantifications of the protein bands were shown after normalization with their respective β-actin levels. Data are presented as means ± SE, n=3. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Inhibition, Expressing

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (2-10 mM, 0-24 h) decreases Hes1 and HRT1 protein levels in HeLa cells. B. NAC treatment (0-10 mM for 6 h or 5 mM for 0-12 h) decreases Hes1 and HRT1 mRNA expression in HeLa cells. The mRNA expression of NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. C. NAC treatment (0-10 mM, 12 h) inhibits Hes1 reporter activity in HeLa cells. The luciferase activity in NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. D. Notch3 siRNA knockdown reduces Hes1 and HRT1 levels in HeLa cells. Protein levels were determined 2 days after siRNA transfection. siCtrl, scramble siRNA; siNotch3, Notch3 siRNA. Protein densitometry quantifications were shown after normalization with β-actin levels. Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Activity Assay, Luciferase, Knockdown, Transfection

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Pre-treatment with a γ-secretase inhibitor, DAPT (20 μM, 30 min), had no effect on NAC-induced (5 mM, 0-12 h) decrease in N3IC protein expression in HeLa cells. B. NAC treatment (5 mM, 0-12 h) did not affect Notch3 mRNA expression in HeLa cells. C. Pre-treatment with NH 4 Cl (25 mM, 1 h), but not lactacystin (10 μM, 30 min), reversed NAC-induced (5 mM, 0-12 h) decrease of N3IC protein levels in HeLa cells. D. NAC treatment did not affect levels of exogenously expressed Notch3 active intracellular domain (N3ICD). HeLa cells were transfected with vectors expressing N3ICD or N3FL for 24 h, followed by treatment with NAC (5 mM, 0-12 h). E. Subcellular analysis of Notch3 protein levels following NAC treatment (5 mM, 6 h) in HeLa cells. Protein levels of N3FL, N3EC and N3IC in cytosolic, nuclear and membrane fractions were determined. Successful fractionation was evidenced by using the marker proteins GAPDH, cyclin B1, and Na + , K + -ATPase. N3FL, N3EC and N3IC denoted Notch3 full length, extracellular domain and intracellular domain, respectively. Protein densitometry quantifications were shown after normalization with β-actin (A, C, D) or their respective cellular compartment markers (E). Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Transfection, Membrane, Fractionation, Marker

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: N3ICD overexpression rescues NAC-induced inhibition of proliferation (A), migration (B), and invasion (C). A. Numbers of EV- and N3ICD-transfected cells were counted at 12-48 h after NAC treatment (0-10 mM, left panel). *, p < 0.05 compared with the EV-transfected cells within the same treatment and time point. B. Results of the wound healing assay (left panels) were expressed as the migration index (the distance migrated relative to the initial scraped gap) and that of EV-transfected cells without NAC treatment was set as 100% (middle panel). C. Cells per field on the insert membrane were imaged (left panels) and counted (middle panel). B and C: *, p < 0.05 compared with no NAC treatment; #, p < 0.05 compared with the EV-transfected cells within the same treatment. Percent rescue (A-C, right panels) after N3ICD expression was calculated by dividing the net change after NAC treatment in N3ICD-transfected cells by that in EV-transfected cells. Notch3 siRNA knockdown inhibits cell proliferation D. , migration E. , and invasion F. as assessed by the same approaches described above. Representative images for migration and invasion were shown. *, p < 0.05 compared with the siCtrl-transfected cells. All data are presented as mean ±SE, n=3. I, the initial seeded cell number. EV, empty vector; N3ICD, Notch3 active intracellular domain; siCtrl, scrambled siRNA; siNotch3, Notch3 siRNA.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Over Expression, Inhibition, Migration, Transfection, Wound Healing Assay, Membrane, Expressing, Knockdown, Plasmid Preparation

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (5 and 10 mM, 0-24 h) decreases N3IC protein levels in HCC1937 cells. Expression of exogenous N3ICD rescues NAC-induced inhibition of proliferation B. , migration C. , and invasion D. , and Notch3 siRNA knockdown inhibits proliferation E. , migration F. , and invasion G. in HCC1937 cells. Quantifications, sample size, statistics, and abbreviations for protein levels, proliferation, migration, and invasion assays were as described in Figure & legends.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Inhibition, Migration, Knockdown