Journal: OncoTargets and therapy

Article Title: Epigallocatechin gallate inhibits the proliferation of colorectal cancer cells by regulating Notch signaling

doi: 10.2147/OTT.S40914

Figure Lengend Snippet: The effect of EGCG on expressions of Notch1, Notch2, HES1 , and JAG1 in the colorectal cancer cells: ( A ) the expression of HES1 ; ( B ) the expression of JAG1 ; ( C ) the expression of Notch1 ; and ( D ) the expression of Notch2 . Notes: The x-axis shows the Lovo, SW480, HT-29, and HCT-8 colorectal cell lines. The y-axis shows the mRNA of the colorectal cell lines after treatment with EGCG. An independent-sample t -test was used to analyze the mRNA levels of the colorectal cell lines. Abbreviation: EGCG, epigallocatechin gallate.

Article Snippet: The primary antibodies used were Abcam Hes1 antibody (ab71559), CST Jagged1 (28H8Rabbit mAb), CST Notch1 (D1E11 XP™ Rabbit mAb), and CST Notch2 (8A1Rabbit mAb) (Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing

Journal: Theranostics

Article Title: Serelaxin alleviates cardiac fibrosis through inhibiting endothelial-to-mesenchymal transition via RXFP1

doi: 10.7150/thno.38640

Figure Lengend Snippet: Serelaxin promotes the preservation of endothelial cell properties in both in vivo and in vitro by regulating candidate genes. (A) Heat map and (B) scatter plot show the genes with altered mRNA expression level in only TGFβ1-treated (x-axis) or in both TGFβ1- and Serelaxin-treated (y-axis) HCAECs. STEAP1, NOTCH1, JAGGED1, RAC1, DSP, GSC, ERBB3, FZD7, GSK3B and TGFB2 were significantly regulated by Serelaxin treatment, which is shown by separation from dot lines (cut-off by 4 folds). (C-D) qPCR analysis showing the expression of candidate genes, which were identified from qPCR array in TGFβ1- and Serelaxin-treated MCECs. Cells without any treatment were used as control. (E) qPCR analysis of the panel of candidate genes in vivo in sham and AAC-operated mice treated with vehicle or Serelaxin. Serelaxin significantly rescued the effects of AAC on all genes except for Gsc and Steap1. Student t-test was used for single comparison and one-way ANOVA with Bonferroni post-hoc analysis was used for multiple group comparisons. Gene expression and associated error bars, representing mean ± SEM n≥3, n.s. no significance, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Rabbit Anti-Mouse Jagged1 , 1:50 , Santa Cruz , SC8303.

Techniques: Preserving, In Vivo, In Vitro, Expressing, Control, Comparison, Gene Expression

Journal: Theranostics

Article Title: Serelaxin alleviates cardiac fibrosis through inhibiting endothelial-to-mesenchymal transition via RXFP1

doi: 10.7150/thno.38640

Figure Lengend Snippet: Serelaxin rescues the Notch1 pathway in AAC-operated hearts. Immunofluorescence staining of (A) Jagged1, (B) Notch1 and (C) NICD in combination with WGA and DAPI in sham and AAC-operated hearts treated with vehicle or Serelaxin. Quantifications showed that protein expressions of (D) Jagged1, (E) Notch1 and (F) NICD were downregulated in AAC-operated hearts and could be restored by Serelaxin. (G) Western blot analysis shows the amount of the soluble form of Jagged1 in the medium of TGFβ1-treated MCECs. Ponceau-S stained membrane picture indicates that an equal amount of total precipitated protein was loaded for both the control and TGFβ1-treated MCECs. Soluble Jagged1 was reduced in TGFβ1-treated MCECs as quantified by densitometry analysis (right panel). Student t-test was used for single comparison and one-way ANOVA with Bonferroni post-hoc analysis was used for multiple group comparisons. Gene expression and associated error bars, representing mean ± SEM, n≥3, n.s. no significance, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Rabbit Anti-Mouse Jagged1 , 1:50 , Santa Cruz , SC8303.

Techniques: Immunofluorescence, Staining, Western Blot, Membrane, Control, Comparison, Gene Expression

Journal: Theranostics

Article Title: Serelaxin alleviates cardiac fibrosis through inhibiting endothelial-to-mesenchymal transition via RXFP1

doi: 10.7150/thno.38640

Figure Lengend Snippet: Antibodies used in Immunofluorescence staining

Article Snippet: Rabbit Anti-Mouse Jagged1 , 1:50 , Santa Cruz , SC8303.

Techniques: Immunofluorescence

Journal: Theranostics

Article Title: Serelaxin alleviates cardiac fibrosis through inhibiting endothelial-to-mesenchymal transition via RXFP1

doi: 10.7150/thno.38640

Figure Lengend Snippet: Primers used in qPCR assay for gene expression analysis

Article Snippet: Rabbit Anti-Mouse Jagged1 , 1:50 , Santa Cruz , SC8303.

Techniques: Gene Expression, Sequencing

Journal: EMBO Molecular Medicine

Article Title: Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain

doi: 10.1002/emmm.201201623

Figure Lengend Snippet: Primary rat astrocytes were cultured in the presence of CM prepared from MB231, 231BrM, CN34 and CN34BrM cells and the expression of JAG1 was measured by qRT-PCR and Western blot (inserted photo). Primary rat astrocytes were cultured with the CM from MB231 or 231BrM, and the expression of JAG1 was measured at various time points by qRT-PCR and Western blot (inserted photo). Immortalized human astrocytes cell line (UC1) was cultured in the presence of CM from MB231 or 231BrM cells and the expression of JAG1was measured by RT-PCR. Primary rat astrocytes were cultured in the presence of CM of MB231 or 231BrM, and the expression of JAG1 and reactive astrocytes marker, GFAP, were examined by immunocytochemical staining. Bar, 100 µm. P values were calculated by a two-tailed Student's t test.

Article Snippet: For frozen section staining, slides fixed with methanol were washed with PBS and blocked by 2%BSA for 1 h. After blocking, the slides were washed again with PBS and incubated with anti-JAG1 rabbit polyclonal antibody (1/200; Cell Signaling Technology), anti-GFAP rabbit polyclonal antibody (FITC conjugated, 1/200; Cell Signaling), primary anti-CD44 conjugated with FITC (1/200; Cell Signaling Technology) and anti-ESA conjugated with Alexa 555(1/200; Cell Signaling Technology) for 1 h at room temperature.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, Marker, Staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain

doi: 10.1002/emmm.201201623

Figure Lengend Snippet: Primary rat astrocytes were cultured in the presence of IL-1β at various doses for 24 h, and the JAG1 expression level was measured by qRT-PCR and Western blot (left panel). Primary rat astrocytes were cultured in the presence of 50 ng/ml of IL-1β, and the JAG1 expression was measured at various time points by qRT-PCR and Western blot (right panel). Primary human astrocytes were cultured in the presence of 50 ng/ml of IL-1β, and the JAG1 expression was measured by qRT-PCR and Western blot. Primary rat astrocytes were cultured in the presence of IL-1β for 24 h, and the expressions of JAG1 and GFAP were examined by immunocytochemical staining. Bar, 50 µm. Primary rat astrocytes were cultured in the presence of CM of 231BrM cells with or without 100 ng/ml IL-1RA for 24 h, and the levels of JAG1 mRNA and protein were examined by qRT-PCR and Western blot (inserted photo). Primary rat astrocytes were cultured in 231BrM CM with or without 10 µg/ml of IL-1β antibody for 24 h, and the expression of JAG1 mRNA was examined by qRT-PCR. Primary rat astrocytes were cultured in the presence or absence of IL-1β (50 ng/ml) with or without the NF-κB inhibitor, PDTC, and the expression of JAG1and P50 were measured by qRT-PCR (left panel) and Western blot (right panel). P values were calculated by a two-tailed Student's t test.

Article Snippet: For frozen section staining, slides fixed with methanol were washed with PBS and blocked by 2%BSA for 1 h. After blocking, the slides were washed again with PBS and incubated with anti-JAG1 rabbit polyclonal antibody (1/200; Cell Signaling Technology), anti-GFAP rabbit polyclonal antibody (FITC conjugated, 1/200; Cell Signaling), primary anti-CD44 conjugated with FITC (1/200; Cell Signaling Technology) and anti-ESA conjugated with Alexa 555(1/200; Cell Signaling Technology) for 1 h at room temperature.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain

doi: 10.1002/emmm.201201623

Figure Lengend Snippet: Rat primary astrocytes with or without knockdown of JAG1 were grown as a monolayer, and 231BrM-GFP cells were cultured alone or on top of the astrocytes in the presence or absence of DAPT (10 µM) for 48 h. NICD expression in cancer cells was then examined by immunocytochemical staining. Bar, 100 µm. 231BrM cells were co-cultured with rat primary astrocytes for the indicated time and the population of CSCs (CD24 − , CD44 + , ESA + ) was measured by FACS. CSCs were isolated from 231BrM cells by MACS and they were co-cultured with primary rat astrocytes, NIH3T3 or mouse brain endothelial cells (Brain ET) for 72 h. Cells were then subjected to FACS analysis using antibodies to CD24, CD44 and ESA. CSCs from 231BrM were co-cultured with rat astrocytes in the presence of various concentrations of DAPT for 72 h followed by FACS analysis using antibodies to CD24, CD44 and ESA. CSCs were isolated from 231BrM/Tet-NICD cells, and they were treated with or without tetracycline to induce NICD for 48 h followed by FACS analysis using antibodies to CD24, CD44 and ESA. P values were calculated by a two-tailed Student's t test.

Article Snippet: For frozen section staining, slides fixed with methanol were washed with PBS and blocked by 2%BSA for 1 h. After blocking, the slides were washed again with PBS and incubated with anti-JAG1 rabbit polyclonal antibody (1/200; Cell Signaling Technology), anti-GFAP rabbit polyclonal antibody (FITC conjugated, 1/200; Cell Signaling), primary anti-CD44 conjugated with FITC (1/200; Cell Signaling Technology) and anti-ESA conjugated with Alexa 555(1/200; Cell Signaling Technology) for 1 h at room temperature.

Techniques: Knockdown, Cell Culture, Expressing, Staining, Isolation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain

doi: 10.1002/emmm.201201623

Figure Lengend Snippet: Primary rat astrocytes with or without knockdown of JAG1 were grown as a monolayer, and 231BrM-GFP cells were cultured alone or on top of the astrocytes for 48 h. GFP + cells were then isolated by FACS, and the expression of HES5 was measured by qRT-PCR. Kaplan–Meier analysis for brain metastasis-free survival of 204 breast cancer patients (GSE12276). Patients were divided into two groups based on the expression status of HES5 in the primary tumour. HES5 mRNA levels in the primary ( n = 5) and brain metastatic samples ( n = 8) of breast cancer patients were examined by Taqman Real time PCR. 231BrM/Tet-NICD cells were cultured in the presence or absence of tetracycline and with or without infection of lenti virus expressing sh-HES5 for 72 h followed by FACS analysis for CSCs population. Mammosphere forming ability was measured in CSCs that were isolated from 231BrM/Tet-NICD cells in the presence or absence of tetracycline and with or without infection of sh-HES5 lenti virus. Representative photos were taken at day 18 (inserted figure). Bar, 200 µm. HES5 was ectopically expressed in 231BrM cells by lenti virus infection, and CSCs population was measured by FACS. The over expression of HES5 in 231BrM was verified by Western blot (inserted figure). CSCs were isolated from primary breast tumour cells that were infected with indicated lenti viruses, and mammosphere forming abilities were measured. Representative photos were taken at day 14 (inserted figure). Bar, 200 µm. Primary breast tumour cells with or without infection of lenti virus expressing HES5 were cultured in a low-attachment plate for 72 h followed by FACS analysis for CSCs population. P values were calculated by a two-tailed Student's t test.

Article Snippet: For frozen section staining, slides fixed with methanol were washed with PBS and blocked by 2%BSA for 1 h. After blocking, the slides were washed again with PBS and incubated with anti-JAG1 rabbit polyclonal antibody (1/200; Cell Signaling Technology), anti-GFAP rabbit polyclonal antibody (FITC conjugated, 1/200; Cell Signaling), primary anti-CD44 conjugated with FITC (1/200; Cell Signaling Technology) and anti-ESA conjugated with Alexa 555(1/200; Cell Signaling Technology) for 1 h at room temperature.

Techniques: Knockdown, Cell Culture, Isolation, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Infection, Virus, Over Expression, Western Blot, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain

doi: 10.1002/emmm.201201623

Figure Lengend Snippet: CSCs or non-CSCs were isolated from 231BrM cells, and a limiting dilution analysis was done by intracardially injecting various numbers of cells into nude mice followed by monitoring the incidence of brain metastasis by BLI. The brain sections of the tumour bearing mice were subjected to immunohistochemical analysis for JAG1 (red, Alexa 555), GFAP (green, FITC) and H&E staining. The astrocyes which express both JAG1 and GFAP are indicated by white arrows. Met, metastasis BP, brain peripheral, Bar, 100 µm. The brain sections of the tumour bearing mice were subjected to immunohistochemical analysis for CD44 (green, FITC), ESA (red, Alexa 555) and H&E staining. Cancer cells expressing both markers are indicated by white arrows. Bars indicate100 µm (left panel) and 50 µm(middle and right panels), respectively. CSCs were prepared from 231BrM, 231BrM/DNMAML and 231BrM/shIL-1, and 5 × 10 4 cells were intracardially injected into nude mice followed by monitoring tumour growth by measuring the total photon flux in the brain. Brian metastasis free survival curve was shown in right panel. CSCs were prepared from MDA231 and MDA231-IL1β, and 10 5 cells were intracardially injected into nude mice followed by measuring the total photon flux in the brain ex vivo at the end point (left panel). The result of brian metastasis-free survival was shown in the right panel. CSCs were prepared from 231BrM cells and 5 × 10 4 cells were intracardially injected to each group of mice ( n = 12) followed by treatment with Compound E or vehicle only followed by measuring the total photon flux in the brain for 32 days. Proposed model for the growth of breast CSCs in the brain. IL-1β secreted from metastatic CSCs up-regulates JAG1 on the reactivated astrocytes which in turn promote self-renewal of CSCs through JAG1-Notch-HES5 axis. P values were calculated by a two-tailed Student's t test. Proposed model for the growth of breast CSCs in the brain. IL-1β secreted from metastatic CSCs up-regulates JAG1 on the reactivated astrocytes which in turn promote self-renewal of CSCs through JAG1-Notch-HES5 axis. p -values were calculated by a two-tailed Student's t test.

Article Snippet: For frozen section staining, slides fixed with methanol were washed with PBS and blocked by 2%BSA for 1 h. After blocking, the slides were washed again with PBS and incubated with anti-JAG1 rabbit polyclonal antibody (1/200; Cell Signaling Technology), anti-GFAP rabbit polyclonal antibody (FITC conjugated, 1/200; Cell Signaling), primary anti-CD44 conjugated with FITC (1/200; Cell Signaling Technology) and anti-ESA conjugated with Alexa 555(1/200; Cell Signaling Technology) for 1 h at room temperature.

Techniques: Isolation, Immunohistochemical staining, Staining, Expressing, Injection, Ex Vivo, Two Tailed Test

Journal: Journal of Diabetes Research

Article Title: The Involvement of Notch1-RBP-Jk/Msx2 Signaling Pathway in Aortic Calcification of Diabetic Nephropathy Rats

doi: 10.1155/2017/8968523

Figure Lengend Snippet: The mRNA expressions of Notch1 (a), RBP-Jk (b), Msx2 (c), and Jagged1 (d) in aortic tissues. Data presented as mean ± standard deviation (SD). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus Nor group; Δ p > 0.05, # p < 0.05, ## p < 0.01, and ### p < 0.001. Nor group: normal controls; DN + VDN group: diabetic nephropathy rats with vitamin D3/nicotine-induced vascular calcification.

Article Snippet: The following primary antibodies were used for immunohistochemical analysis: monoclonal rabbit anti-rat RBP-Jk, Msx2, and N1-ICD (Shanghai Bioworld Tech Inc., China); monoclonal rabbit anti-rat Jagged1 and Runx2 (Beijing BioTech Inc., China); monoclonal rabbit anti-rat Notch1 (Wuhan Proteintech Group Inc., China); and monoclonal mouse anti-rat α -SMA (Wuhan Boster Bio-Tech., Ltd, China).

Techniques: Standard Deviation

Journal: Journal of Diabetes Research

Article Title: The Involvement of Notch1-RBP-Jk/Msx2 Signaling Pathway in Aortic Calcification of Diabetic Nephropathy Rats

doi: 10.1155/2017/8968523

Figure Lengend Snippet: Detection of Notch1, RBP-Jk, Msx2, Jagged1, and N1-ICD levels by immunohistochemical analysis. The representative images and quantitative analysis of Notch1 (a-b), RBP-Jk (c-d), Msx2 (e-f), Jagged1 (g-h), and N1-ICD (i-j) in aortic tissues. The left panel of histological images: Nor group; right panel: DN + VDN group at 8, 12 , and 16 weeks, respectively. Data presented as mean ± standard deviation (SD). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus Nor group; Δ p > 0.05, # p < 0.05, ## p < 0.01, and ### p < 0.001. Nor group: normal controls; DN + VDN group: diabetic nephropathy rats with vitamin D3/nicotine-induced vascular calcification.

Article Snippet: The following primary antibodies were used for immunohistochemical analysis: monoclonal rabbit anti-rat RBP-Jk, Msx2, and N1-ICD (Shanghai Bioworld Tech Inc., China); monoclonal rabbit anti-rat Jagged1 and Runx2 (Beijing BioTech Inc., China); monoclonal rabbit anti-rat Notch1 (Wuhan Proteintech Group Inc., China); and monoclonal mouse anti-rat α -SMA (Wuhan Boster Bio-Tech., Ltd, China).

Techniques: Immunohistochemical staining, Standard Deviation