primary antibodies for notch3  (Cell Signaling Technology Inc)

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: Primary antibodies for Notch3 (5276S), E-cadherin (3195S), Vimentin (5471S), and β-actin (3700S) were purchased from Cell Signaling Technology (Danvers, USA).

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: Primary antibodies for Notch3 (5276S), E-cadherin (3195S), Vimentin (5471S), and β-actin (3700S) were purchased from Cell Signaling Technology (Danvers, USA).

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: Primary antibodies for Notch3 (5276S), E-cadherin (3195S), Vimentin (5471S), and β-actin (3700S) were purchased from Cell Signaling Technology (Danvers, USA).

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: Primary antibodies for Notch3 (5276S), E-cadherin (3195S), Vimentin (5471S), and β-actin (3700S) were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy

doi: 10.1101/2023.07.10.548456

Figure Lengend Snippet: a , Single-cell RNA sequencing UMAP of 30,934 transcriptomes from human meningioma samples with loss of chromosome 22q showing tumor cell states and microenvironment cell types. b , UMAP showing single-cell RNA sequencing of human meningiomas shaded by chromosome 22q status. c , Dot plot showing expression of NOTCH receptors ( NOTCH1, NOTCH2, NOTCH3, NOTCH3) , NOTCH ligands ( JAG1, JAG2, DLL1, DLL3, DLL4, DLK2, FN1) , mural cell markers ( PDGFRB, RGS5, CSPG4, ACTA2, ABCC9, ANGPT2, GJA4, CD248, COL9A3, SDC2, CLU, SERPING1 ), cancer stem-cell marker ( NOTCH3, THY1 ), cell proliferation markers ( MKI67, TOP2A, AURKB ), and endothelial cells markers ( ADM, PDGFD, CD3, VWF, CLDN5, PECAM1, KDR, FLT1, PRCP, MFSD2A, CXCL12, TIE1 ) across meningioma mural (C7, C11, C12) or endothelial cells (C8) from a. d , Inference of NOTCH signaling network in human meningiomas using single-cell RNA sequencing cell-cell communication analysis. e , Magnetic resonance imaging (MRI, left) or H&E images (right) of spontaneous dog meningiomas. Scale bars, 100µm. f , Transcriptomic concordance of human meningioma single-cell cluster identities from a projected on single-cell RNA sequencing UMAP of 40,525 transcriptomes from dog meningioma samples showing NOTCH3+ meningioma mural cells and proliferating meningioma cells are conserved across human and dog meningiomas. g , IHC for NOTCH3 across histological grades of human (top) or dog (bottom) meningiomas. Representative of n=3-10 meningiomas per grade. Scale bars, 100µm. h , IF for NOTCH3 and the mural cell marker SMA across histological grades of human meningiomas. DAPI marks DNA. Representative of n=10 meningiomas per grade. Scale bars, 10µm. i , Quantification of NOTCH3 or the NOTCH3 target gene HEY1 across meningioma grades using RNA sequencing of n=502 human meningiomas. TPM, transcripts per million. Lines represent means and error bars represent standard error of means. ANOVA.

Article Snippet: Primary antibodies recognizing NOTCH3 (5276, Cell Signaling, 1:1000) or GAPDH (8245, Abcam, 1:5000) and secondary antibodies recognizing mouse (7076, Cell Signaling, 1:2000) or rabbit (7074, Cell Signaling, 1:2000) epitope were used.

Techniques: RNA Sequencing, Expressing, Marker, Magnetic Resonance Imaging

Journal: bioRxiv

Article Title: NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy

doi: 10.1101/2023.07.10.548456

Figure Lengend Snippet: a , IHC for NOTCH3 in the adult human meninges showing expression is restricted to mural cells. Representative of n=3 biological replicates. Scale bars, 100µm and 10µm (insert). b , IF for NOTCH3 and the mural cell marker SMA in 3 adult human meningeal samples showing NOTCH3 is expressed in mural cells adjacent to smooth muscle cells in the meninges. DAPI marks DNA. Scale bar, 10µm. c , Experimental design for in vivo lineage tracing of NOTCH3+ mural cells during meningeal development ( in utero recombination) or homeostasis (postnatal recombination). TAM, tamoxifen. d , Confocal microscopy of whole mount mouse convexity meningeal samples at P7, P30, or P90 after in utero recombination of the ROSA mT/mG allele showing NOTCH3 cells (green) are restricted to the perivascular niche during meningeal development. Representative of n=3 biological replicates per timepoint. DAPI marks DNA. Scale bar, 10µm. e , Confocal microscopy of whole mount mouse convexity meningeal samples at P30 or P90 after postnatal recombination of the ROSA mT/mG allele showing NOTCH3+ cells (green) are restricted to the perivascular niche during meningeal homeostasis. Representative of n=3 biological replicates per timepoint. DAPI marks DNA. Scale bar, 10µm. f , Experimental design for in vivo biallelic inactivation of Nf2 in NOTCH3+ cells during meningeal development (E16.5) or homeostasis (P30). Mice were monitored for 1 year after Nf2 inactivation. g , Coronal H&E images of 300µm decalcified mouse skull sections 1 year after treatment of mice with TAM. No gross tumors were identified, but insets show Nf2 inactivation in NOTCH3+ cells is associated with meningeal hyperproliferation after either in utero (E16.5) or postnatal (P30) treatment with TAM. Representative of n=5-8 biological replicates per condition. Scale bars 1mm.

Article Snippet: Primary antibodies recognizing NOTCH3 (5276, Cell Signaling, 1:1000) or GAPDH (8245, Abcam, 1:5000) and secondary antibodies recognizing mouse (7076, Cell Signaling, 1:2000) or rabbit (7074, Cell Signaling, 1:2000) epitope were used.

Techniques: Expressing, Marker, In Vivo, In Utero, Confocal Microscopy

Journal: bioRxiv

Article Title: NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy

doi: 10.1101/2023.07.10.548456

Figure Lengend Snippet: a , Deconvolution of NOTCH3+ meningioma mural cells from using human meningiomas with paired RNA sequencing and DNA methylation profiling (n=502). ANOVA. b , Immunoblots showing NOTCH3 is expressed in CH-157MN and IOMM-Lee Immune-enriched meningioma cell lines. c , In vivo tumor initiating capacity of CH-157MN meningioma cells in NU/NU mice ± αNRR3 IP injection 2 times per week. Denominators indicate number of mice at each time point. Numerators indicate number of mice with tumors at each time point. d , QPCR for the NOTCH3 target gene HEY1 from meningioma xenografts ± αNRR3 treatment for 2 weeks. Student’s t test. e , IHC for Ki-67 in meningioma xenografts showing αNRR3 blocks meningioma cell proliferation. Representative of n=3 xenografts per condition. Scale bar, 100µm. f , CH-157MN meningioma xenograft growth (left, student’s t tests) or survival (log-rank test). Arrows indicate initiation of bi-weekly treatment with the indicated therapy, which continued until death. g , IOMM-Lee meningioma xenograft growth (left, student’s t tests) or survival (log-rank test). Arrows as in f. h , QPCR for NOTCH3 or HEY1 in CH-157MN meningioma cells ± stable expression of empty vector (EV) or NOTCH3 ICD . Student’s t tests. i , IF quantification of the stem cell marker PTPRZ1 in CH-157MN meningioma cells. Student’s t test. j , Clonogenic in vitro growth of CH-157MN meningioma cells after 2 weeks. Student’s t test. k , In vivo tumor initiating capacity of CH-157MN meningioma cells ± EV or NOTCH3 ICD over limiting dilutions. Numerator and denominator as in c. l , CH-157MN meningioma xenograft growth (left, student’s t tests) or survival (log-rank test). m , Images of heterotopic meningioma xenografts showing macroscopic necrosis and ulceration in EV meningiomas. Representative of n=7-9 xenografts per condition. n , H&E low and high (box) magnification images of meningioma xenografts showing microscopic necrosis in EV meningiomas. Representative of n=3 xenografts per condition. Scale bars, 100µm. o , IHC for endothelia markers in meningioma xenografts showing NOTCH3 ICD induces meningioma angiogenesis. Representative of n=3 xenografts per condition. Scale bars, 100µm. Lines represent means and error bars represent standard error of means. **p≤0.01, ***p≤0.0001.

Article Snippet: Primary antibodies recognizing NOTCH3 (5276, Cell Signaling, 1:1000) or GAPDH (8245, Abcam, 1:5000) and secondary antibodies recognizing mouse (7076, Cell Signaling, 1:2000) or rabbit (7074, Cell Signaling, 1:2000) epitope were used.

Techniques: RNA Sequencing, DNA Methylation Assay, Western Blot, In Vivo, Injection, Expressing, Plasmid Preparation, Marker, In Vitro

Journal: bioRxiv

Article Title: NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy

doi: 10.1101/2023.07.10.548456

Figure Lengend Snippet: a , Network of gene circuits distinguishing recurrent (n=99) from primary (n=403) human meningiomas using RNA sequencing. Nodes represent pathways and edges represent shared genes between pathways (p≤0.01, FDR≤0.01). Red nodes are enriched and blue nodes are suppressed in recurrent versus primary meningiomas. b , IHC for Ki-67 in recurrent (n=53) versus primary (n=123) meningiomas, or RNA sequencing of recurrent (n=99) versus primary (n=403) meningiomas for deconvolution of NOTCH3+ meningioma mural cells from Fig. 1a or quantification of NOTCH3 or HEY1 expression. TPM, transcripts per million. ANOVA. c , Multiplexed seqIF microscopy showing human meningioma recurrence after radiotherapy (RT) is associated with increased NOTCH3 and Ki-67. Many NOTCH3+ cells also express the interferon and innate immune regulators STING and pSTAT3. CD31 marks pericytes, COL1A marks fibroblasts, SSTR2A marks meningioma cells, and DAPI marks DNA. Representative of n=4 pairs of patient-matched primary and recurrent meningiomas. Scale bar, 100µm. d , CH-157MN meningioma xenograft growth (left and middle, student’s t tests) or survival (log-rank test) after expression of empty vector (EV) or NOTCH3 ICD ± RT showing NOTCH3 drives resistance to RT. Arrows indicate RT treatments (2Gy x 5 daily fractions). Xenografts from all arms were isolated for single-cell RNA-sequencing 1 day after completing RT (early) or once median survival was reached in the EV + RT arm (late). e , Single-cell RNA sequencing UMAP of 152,464 meningioma xenograft human cell transcriptomes showing tumor cell states ± αNRR3 treatment for 2 weeks as in Fig. 3f or ± NOTCH3 ICD ± RT as in d. f , UMAP showing single-cell RNA sequencing of meningioma xenograft human cells shaded by experimental condition or phase of the cell cycle. g , Analysis of C2 G2M/S phase meningioma xenograft human cells in control versus NOTCH3 ICD versus αNRR3 conditions showing NOTCH3 drives meningioma cell proliferation. Colors as in f . Student’s t tests. h , Cell cycle analysis across all clusters of meningioma xenograft human cells ± NOTCH3 ICD ± RT showing NOTCH3 sustains cell proliferation through G2M and S phase despite RT. Student’s t test. i , Meningioma xenograft growth (left, student’s t tests) or survival (log-rank test) after treatment with RT as in d ± αNRR3 as in Fig. 3f. αNRR3 treatment was initiated on the first day of radiotherapy and continued until death. Lines represent means and error bars represent standard error of means. *p<0.05, **p≤0.01.

Article Snippet: Primary antibodies recognizing NOTCH3 (5276, Cell Signaling, 1:1000) or GAPDH (8245, Abcam, 1:5000) and secondary antibodies recognizing mouse (7076, Cell Signaling, 1:2000) or rabbit (7074, Cell Signaling, 1:2000) epitope were used.

Techniques: RNA Sequencing, Expressing, Microscopy, Plasmid Preparation, Isolation, Control, Cell Cycle Assay