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

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: Journal of Musculoskeletal & Neuronal Interactions

Article Title: MiRNA-206 Affects the Recovery of Sciatic Function by Stimulating BDNF Activity through the Down-regulation of Notch3 Expression

doi:

Figure Lengend Snippet: Prediction of target molecule for miRNA-206. Note: A, Starbase V2.0 online database prediction for the binding site of mirNA-206 at the 3 ’ UTR of Notch3; B, Dual luciferase reporter gene assay for the regulation of miRNA-206 on Notch3; C, qRT-PCR for the regulation of miRNA-206 on Notch3. Notch3 wild type: Notch3-3 ′ UTR WT; Notch3 mutant: Notch3-3 ’ UTR Mut; *P < 0.05; **P < 0.01, N=5.

Article Snippet: The protein was then retransferred to PVDF membrane (Millipore, MA, USA, IPFL00010), and then closed with 5% skim milk and incubated with the following primary antibodies at 4°C: Mouse anti-Notch3 (1:1000, Millipore, MA, USA, APREST76106), Mouse anti-BNDF (1:1000, Sigma-Aldrich, USA, GF029) and Rabbit anti-GAPDH (1:1000, Millipore, MA, USA, G5262).

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Mutagenesis

Journal: Journal of Musculoskeletal & Neuronal Interactions

Article Title: MiRNA-206 Affects the Recovery of Sciatic Function by Stimulating BDNF Activity through the Down-regulation of Notch3 Expression

doi:

Figure Lengend Snippet: Western blotting for expressions of Notch3 and BDNF. Note: A, Protein expression of Notch3 in different transected nerve groups; B, mRNA expression of Notch3 in different transected nerve groups; C, Detection on Notch3 for BDNF protein expression; D, Detection on Notch3 for mRNA expression of BDNF. Notch3 over-expression control group: Notch3-ctrl; Notch3 over-expression group: Notch3 mimic; Sham: Nerve exposed group not operated; Model: Denervation group; PAG: Nerve partial anastomosis group; AG: Nerve anastomosis group. *P < 0.05; **P < 0.01, N=5.

Article Snippet: The protein was then retransferred to PVDF membrane (Millipore, MA, USA, IPFL00010), and then closed with 5% skim milk and incubated with the following primary antibodies at 4°C: Mouse anti-Notch3 (1:1000, Millipore, MA, USA, APREST76106), Mouse anti-BNDF (1:1000, Sigma-Aldrich, USA, GF029) and Rabbit anti-GAPDH (1:1000, Millipore, MA, USA, G5262).

Techniques: Western Blot, Expressing, Over Expression

Journal: Journal of Musculoskeletal & Neuronal Interactions

Article Title: MiRNA-206 Affects the Recovery of Sciatic Function by Stimulating BDNF Activity through the Down-regulation of Notch3 Expression

doi:

Figure Lengend Snippet: miRNA-206 targeting Notch3 affects the recovery of neural function. Note: A: Western blotting for miRNA-206 regulating BDNF expression by Notch3; B-D: qRT-PCR for miRNA-206 regulating BDNF expression by Notch3; miRNA-206 over-expression control group: miRNA-206-ctrl, miRNA-206 over-expression group: miR-206 mimic; *P < 0.05; **P < 0.01, N=5.

Article Snippet: The protein was then retransferred to PVDF membrane (Millipore, MA, USA, IPFL00010), and then closed with 5% skim milk and incubated with the following primary antibodies at 4°C: Mouse anti-Notch3 (1:1000, Millipore, MA, USA, APREST76106), Mouse anti-BNDF (1:1000, Sigma-Aldrich, USA, GF029) and Rabbit anti-GAPDH (1:1000, Millipore, MA, USA, G5262).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Over Expression

Journal: Journal of Musculoskeletal & Neuronal Interactions

Article Title: MiRNA-206 Affects the Recovery of Sciatic Function by Stimulating BDNF Activity through the Down-regulation of Notch3 Expression

doi:

Figure Lengend Snippet: Plot of recovery mechanism of miRNA-206 affecting the recovery of neural function by targeting Notch3. Note: Over-expression of miRNA-206 inhibited Notch3 expression and activated the BDNF activity, which ultimately caused the recovery of traumatic sciatic function.

Article Snippet: The protein was then retransferred to PVDF membrane (Millipore, MA, USA, IPFL00010), and then closed with 5% skim milk and incubated with the following primary antibodies at 4°C: Mouse anti-Notch3 (1:1000, Millipore, MA, USA, APREST76106), Mouse anti-BNDF (1:1000, Sigma-Aldrich, USA, GF029) and Rabbit anti-GAPDH (1:1000, Millipore, MA, USA, G5262).

Techniques: Over Expression, Expressing, Activity Assay