Journal: BMC cancer

Article Title: Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

doi: 10.1186/s12885-023-11746-w

Figure Lengend Snippet: Fig. 3 Expression of STAT5A is positively associated with Notch3 in breast cancer. a In TCGA (Cell 2015), the expression of STAT5A was positively associated with Notch3 in patient tissues. b Notch3 and STAT5A mRNA levels in different breast cancer cell lines. c STAT5A protein expression was positively associated with Notch3 in different breast cancer cell lines. d Overexpression of Notch3 promoted expression of STAT5A and its active form, p-STAT5, at the protein level in BT549 cells. e Overexpression of Notch3 promoted the expression of STAT5A and its active form, p-STAT5, at the protein level in MCF-7 cells. f In MCF-7 cells, knockdown of Notch3 decreased STAT5A and p-STAT5A at the protein level. g Overexpression of Notch3 increased the mRNA level of STAT5A in BT549 cells. h Overexpression of Notch3 increased STAT5A mRNA levels in MCF-7 cells. i The mRNA level of STAT5A was suppressed by siNotch3

Article Snippet: Cell lysates were collected from MCF-7 cells at 80% confluence in a 100-mm dish and incubated with anti-Notch3 antibody or normal rabbit IgG (CST 2729) as control.

Techniques: Expressing, Over Expression, Knockdown

Journal: BMC cancer

Article Title: Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

doi: 10.1186/s12885-023-11746-w

Figure Lengend Snippet: Fig. 4 Notch3 directly binds to the STAT5A promoter and activates STAT5A expression. a Potential CSL binding sites of Notch3 on the STAT5A promoter, and the adjacent region, lacking Notch3 binding sites, as the negative control, and the construction strategy of luciferase reporter genes driven by wildtype and CSL-mutant STAT5A promoters. b ChIP results showing that Notch3 directly binds to the STAT5A-2/3 region on the promoter. c In MCF-7 cells, the luciferase activity of pGL3-STAT5A-pro-luc-E was suppressed by loss of Notch3 on the promoter region of STAT5A in a dose-dependent manner. d In MCF-7 cells, the luciferase activity of pGL3-STAT5A-pro-23-luc-E was suppressed by knockdown of Notch3 in a dose-dependent manner. The STAT5A promoter, lacking Notch3 binding sites, was not regulated by knockdown of Notch3. e In BT549 cells, STAT5A promoter-driven luciferase activity was activated by overexpression of Notch3 in a dose-dependent manner. f In BT549 cells, STAT5A promoter-driven luciferase activity was activated by the binding of Notch3 on the STAT5A promoter in a dose-dependent manner. The STAT5A promoter, lacking Notch3 binding sites, was not regulated by the overexpression of Notch3

Article Snippet: Cell lysates were collected from MCF-7 cells at 80% confluence in a 100-mm dish and incubated with anti-Notch3 antibody or normal rabbit IgG (CST 2729) as control.

Techniques: Expressing, Binding Assay, Negative Control, Luciferase, Mutagenesis, Activity Assay, Knockdown, Over Expression

Journal: BMC cancer

Article Title: Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

doi: 10.1186/s12885-023-11746-w

Figure Lengend Snippet: Fig. 5 Notch3-mediated suppression of metastasis can be reversed by STAT5A siRNA in breast cancer cells and high Notch3 and STAT5A expression predicts better prognosis in patients with breast cancer. a Suppressive effect of Notch3 on wound healing was reversed by suppressing STAT5A expression in BT549 cells. b In transwell assays, Notch3-induced decreased mobility of BT549 cells was rescued by knockdown of STAT5A. c Enhancement of wound healing by siNotch3#1 was reversed by overexpression of STAT5A in MCF-7 cells. d Low expression of Notch3 predicted poor recurrence-free survival in patients with breast cancer. As well as, breast cancer patients with low STAT5A levels showed poor recurrence-free survival. e Low expression of Notch3 predicted poor overall survival in patients with breast cancer. And breast cancer patients with low STAT5A levels showed poor overall survival

Article Snippet: Cell lysates were collected from MCF-7 cells at 80% confluence in a 100-mm dish and incubated with anti-Notch3 antibody or normal rabbit IgG (CST 2729) as control.

Techniques: Expressing, Knockdown, Over Expression

Journal: BMC cancer

Article Title: Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

doi: 10.1186/s12885-023-11746-w

Figure Lengend Snippet: Fig. 6 Proposed model of how Notch3 upregulate STAT5A in breast cancer. Notch3 is cleavaged twice to form N3ICD. In the cell nucleus, N3ICD forms a complex with CSL, which initiates transcription of STAT5A. Prolactin(Prl) binding to the receptor(PrlR) results in receptor dimerization and autophosphorylation of the receptor-associated JAK. Then, JAK phosphorylates the receptor, thereby creating docking sites for Src homology 2 (SH2) domain proteins, such as STAT5A. Subsequently, Jak2 phosphorylates the STAT5A to form an active dimer

Article Snippet: Cell lysates were collected from MCF-7 cells at 80% confluence in a 100-mm dish and incubated with anti-Notch3 antibody or normal rabbit IgG (CST 2729) as control.

Techniques: Binding Assay

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