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: International Journal of Oncology

Article Title: Regulation of gliomagenesis and stemness through acid sensor ASIC1a

doi: 10.3892/ijo.2021.5262

Figure Lengend Snippet: Notch signaling is negatively associated with ASIC1a in glioblastoma multiforme. (A-C) The glioma cells were transfected with shASIC1a and control followed by assaying protein expression of ASIC1a, active form of Notch1, Notch2, Notch3, Notch4, and Notch target survivin, as well as GSC markers CD133 and ALDH1 by western blotting in (A) A172, (B) U87MG, and (C) PDX-L12 cells. (D-F) Subsequently, the glioma cells (D) A172, (E) U87MG and (F) PDX-L12 were transfected with ASIC1a-FLAG followed by assaying protein expression of ASIC1a, active form of Notch1, Notch2, Notch3, Notch4 and Notch target survivin, as well as GSC markers CD133 and ALDH1 by western blotting. * P<0.05 and ** P<0.01. ASIC1a, acid-sensing ion channel 1a; sh, short hairpin; GSC, glioblastoma stem cells; ALDH1, aldehyde dehydrogenase 1.

Article Snippet: Rabbit polyclonal anti-Notch3 antibody (product code ab60087) was purchased from Abcam.

Techniques: Transfection, Expressing, Western Blot

Journal: NPJ Breast Cancer

Article Title: Unique cellular protrusions mediate breast cancer cell migration by tethering to osteogenic cells

doi: 10.1038/s41523-020-00183-8

Figure Lengend Snippet: a Ibidi silicone removable chambers were used to quantify migration by breast cancer cells in monoculture, in co-culture with osteoblasts in adjacent chambers, or in direct admixture in the same chamber. b To distinguish cell types, fluorescently labeled breast cancer cells were co-cultured with unlabeled osteoblasts (OBs), and imaged with the Incucyte S3. Scale bar is 100 µm. c Dot plot showing the number of cells escaped from their original enclosure per field for MCF-7 alone, in co-culture with OBs in adjacent regions, or in co-culture with osteoblasts in the same region after 48 h. P -value determined by students t -test (two-tailed). Each dot represents a biological replicate, an average of three technical replicates. d Dot plot shows the number of cells escaped per field for MCF-7 alone, and in co-culture with OBs (represented by hFOB1.19 cells), pre-osteoblasts (Pre-OB represented by MC3T3-E1 cells), human mesenchymal stem cells (MSCs), and osteoclasts (OC RANK-L differentiated Raw 264.7 cells) after 48 h. Each dot represents a technical replicate. d Dot plot shows the number of cells escaped per field for mesenchymal MDA-MB-231 alone and with osteoblasts, as well as MDA-MB-231 E-cadherin expressing subline SCP-28 alone and with osteoblasts after 48 h. Each dot represents a technical replicate. e Dot plot shows the combined data for cells escaped per field for various E-cadherin expressing breast cancer cell lines alone and with osteoblasts. For contrast MDA-MB-231, which lacks E-cadherin expression, is also shown (not included in statistical comparison). P -value determined by students paired t -test (two-tailed). Each dot represents a biological replicate, an average of at least three technical replicates. f Box plot shows the transwell migration (a diagram shown in the left) of MCF-7 cells toward a gradient (0, 1, and 5 µM) of Bone Morphogenetic Protein-2 (BMP-2) with or without admixture of OBs. Each dot represents a biological replicate generated from an independent experiment average of three technical replicates. For c – f error bars indicate standard deviation. (For g ), the lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge. Data beyond the end of the whiskers are called “outlying” points and are plotted individually. P -value as determined by analysis of covariance (ANCOVA) as elaborated in “Methods”.

Article Snippet: Commercially available monoclonal antibodies (mAb) or polyclonal antibodies (pAb) were used for Cofilin (rabbit pAb, Abcam ab42824), GFP (chicken pAb, Abcam ab13970), E-cadherin (rabbit mAb, Cell Signaling Technologies 3195S), N-Cadherin (mouse mAb, Abcam ab98952, Notch3 (rabbit pAb, Abcam ab23426), Cx43 (rabbit pAb, Sigma C6219), Myosin X (rabbit pAb, Novus, 22430002)) and ALP (rabbit mAb, Abcam ab108337).

Techniques: Migration, Co-Culture Assay, Labeling, Cell Culture, Two Tailed Test, Expressing, Generated, Standard Deviation, Whisker Assay

Journal: NPJ Breast Cancer

Article Title: Unique cellular protrusions mediate breast cancer cell migration by tethering to osteogenic cells

doi: 10.1038/s41523-020-00183-8

Figure Lengend Snippet: a MCF-7 E-cadherin (green) expressed where DSLS contacts osteoblast, here visible with phalloidin staining (red). Scale bars are 10 (left), and 5 µm (right), respectively. b MCF-7 E-cadherin (green) binding osteoblast N-cadherin (red) where DSLS contacts osteoblast. Scale bars are 5 (left), 1 µm (right), respectively. c Osteoblast gap junction protein connexin 43 (Cx43 – red) at the site of MCF-7 (green) DSLS contact. Scale bars are 5 (left), 1 (upper right), and 1 µm (lower right), respectively. d Gap junction permeable dye calcein red traversing from MCF-7 to osteoblast via DSLS. Scale bar is 15 µm. e Flow cytometry quantification of calcein dye transfer. The first panel shows un-transfected MCF-7 loaded with calcein red dye. The second shows GFP-labeled osteoblasts. The third shows the co-culture of the two cells after 18 h. f Box plots showing the 2D migration cells through the membrane per field for MCF-7 and AT3 cells treated with 10 µM CBX gap junction inhibitor with and without osteoblasts. Each dot represents a biological replicate, an average of three technical replicates. g Box plots showing the transwell migration cells through the membrane per field for MCF-7 cells treated with 10 µM CBX gap junction inhibitor with and without osteoblasts. Each dot represents a biological replicate, an average of three technical replicates. For box plots, the lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge. Data beyond the end of the whiskers are called “outlying” points and are plotted individually. P -value as determined by either ANOVA ( f ) or ANCOVA ( g ) as elaborated in “Methods”.

Article Snippet: Commercially available monoclonal antibodies (mAb) or polyclonal antibodies (pAb) were used for Cofilin (rabbit pAb, Abcam ab42824), GFP (chicken pAb, Abcam ab13970), E-cadherin (rabbit mAb, Cell Signaling Technologies 3195S), N-Cadherin (mouse mAb, Abcam ab98952, Notch3 (rabbit pAb, Abcam ab23426), Cx43 (rabbit pAb, Sigma C6219), Myosin X (rabbit pAb, Novus, 22430002)) and ALP (rabbit mAb, Abcam ab108337).

Techniques: Staining, Binding Assay, Flow Cytometry, Transfection, Labeling, Co-Culture Assay, Migration, Whisker Assay