Background: Line of HIF-1α / Notch signaling regulate cell proliferation, apoptosis, and metabolism of the intervertebral disc (IVD) and is involved in disc degeneration. The nucleus pulposus (NP) is an important structure adjacent to the IVD. However, the role of HIF-1α / Notch signaling pathway in NP cells obtained from patients with different Modic changes (MC) is still unclear. The purpose of this study was to investigate the role of HIF-1α and components of the Notch pathway in NP obtained from patients with various MC.
Methods: A total of 85 NP tissue samples collected from patients who underwent diskectomy for the treatment of low back pain. NP network were divided into four groups based on the adjacent endplate degeneration, namely, MC I, II, III, and negative MC group. Expression of HIF-1α and Notch-related component is measured and compared.
Results: The expression of HIF-1α, Notch1 and Notch2 gradually increased in the first MC and MC II group than in the negative group MC. HIF-1α and Notch-related components are rarely detected in MC group III. Conclusion: The expression of HIF-1α / Notch increased in cells of patients with NP MC MC I and II. HIF-1α and components related to the potential biomarkers Notch and HIF-1α / Notch signaling pathway may serve as a promising therapeutic target for disc degeneration in patients with MC.
Correlation between the HIF-1α/Notch signaling pathway and Modic changes in nucleus pulposus cells isolated from patients with low back pain
Targeting the Notch signaling pathway and TGF-β to prevent retinal fibrosis in vitro and in vivo
Rationale: Notch and transforming growth factor-β (TGFβ) two signaling pathways that control intracellular mechanisms of fibrosis in general, but if they play a major role in retinal fibrosis is less clear. Here we learn how these two signal pathways regulate cell Müller-dominated retinal fibrosis in vitro and in vivo. Methods: Human cells MIO-M1 Müller and TGFβ1 treated with Notch ligands, either alone or in combination. Western blot was conducted to study the changes protease γ-Secretase, Notch downstream effectors, endogenous TGFβ1, Smad3 phosphorylated (p-Smad3) and the extracellular matrix (ECM) proteins.
We also studied the effects RO4929097, a selective inhibitor of γ-Secretase, the ECM protein expression after ligand stimulation. Müller cell viability and cytotoxicity was studied by AlamarBlue to test the cytotoxicity of lactate. Finally, we studied the changes in Notch and TGFβ signaling and test the effect of an intravitreal injection of Notch pathway inhibitors RO4929097 on retinal fibrosis resulting from sodium iodate (NaIO3) -induced retinal injury in rats.
We also studied the security of RO4929097 intravitreal injection of normal mice. Results: Treatment of Muller cells with protease deregulated Notch ligand and Notch γ-Secretase downstream effector, with an increased expression of endogenous TGFβ1, TGFβ receptor and p-Smad3. TGFβ1 regulated expression of proteins associated with both signal paths in the same way. Notch ligands and TGFβ1 have an additive effect on ECM protein overexpression in Müller cells were inhibited by RO4929097. Notch and TGFβ ligand stimulates proliferation of Müller cells were inhibited by RO4929097 without damaging the cells. NaIO3-induced injury to the retina enabled both Notch and TGFβ signaling pathway in vivo intravitreal injection RO4929097 prevented gliosis Müller cells and inhibit excessive ECM proteins in this murine model.
Human Notch Signaling Pathway Notch1/CSL Reporter Cell Line-HEK293
Description: Human Notch Signaling Pathway Notch1/CSL Reporter Cell Line is derived from HEK293. Application: • Monitor Notch signaling pathway activity.• Screen for activators or inhibitors of the Notch signaling pathway.
Description: The Notch Pathway Reporter kit is designed for monitoring the activity of Notch signaling pathway in the cultured cells. The kit contains the transfection-ready expression vector for Notch1 with the entire extracellular domain deleted (Notch1DE). Inside the cells, the Notch1DE can be cleaved by -secretase and active Notch1 NICD is released to nucleus. The kit also contains a CSL (CBF1/RBP-J) luciferase reporter vector, which is a Notch pathway-responsive reporter. This reporter contains a firefly luciferase gene under the control of multimerized CSL responsive element upstream of a minimal promoter. The CSL (CBF1/RBP-J) reporter is premixed with constitutively expressing Renilla (sea pansy) luciferase vector, which serves as an internal positive control for transfection efficiency._x000D_The kit also includes a non-inducible firefly luciferase vector premixed with constitutivelyexpressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, but without any additional response elements. The negative control is critical for determining pathway specific effects and background luciferase activity.
Description: The Notch CSL Reporter andndash; HEK293 cell line contains the firefly luciferase gene under the control of Notch-response elements (CSL responsive elements) alone with expression construct for Notch1 E (NOTCH1 that has a deletion of the entire extracellular domain) stably integrated into HEK293 cells. Inside the cells, the Notch1 E can be cleaved by andgamma;-secretase. The active Notch1 NICD is released to nucleus and induces the constitutive expression of luciferase reporter. The cell line is validated for the inhibition of the expression of luciferase reporter using a known inhibitor of the Notch signaling pathway.
Notch Signaling Pathway Notch1/CSL Reporter - HEK293 Cell Line
Description: The Notch CSL Reporter - HEK293 cell line contains the firefly luciferase gene under the control of Notch-response elements (CSL responsive elements) alone with expression construct for Notch1 E (NOTCH1 that has a deletion of the entire extracellular domain) stably integrated into HEK293 cells. Inside the cells, the Notch1 E can be cleaved by γ-secretase. The active Notch1 NICD is released to nucleus and induces the constitutive expression of luciferase reporter. The cell line is validated for the inhibition of the expression of luciferase reporter using a known inhibitor of the Notch signaling pathway.
Description: The Notch1/CSL Transient Packis designed to provide the tools necessary for transiently transfecting and monitoring the activity of the Notch signaling pathway in HEK293 cultured cells. The kit contains transfection-ready vectors containing firefly luciferase as a Notch pathway-responsive reporter and constitutively expressing Renilla luciferase as a transfection control. It also includes the Dual Luciferase detection reagents to detect both luciferase activities and specialized medium for growing and assaying HEK293 cells._x000D_The key to the Notch1/CSL Transient Pack is the expression vector for NOTCH1 that has a deletion of the entire extracellular domain (Notch1DE). Inside the cells, the NOTCH1 DE can be cleaved by γ-secretase and active NOTCH1 NICD is released into the nucleus. The kit also contains CSL (CBF1/RBP-Jk) luciferase reporter vector, which is a Notch pathway-responsive reporter. This reporter contains the firefly luciferase gene under the control of multimerized CSL responsive elements upstream of a minimal promoter. The CSL (CBF1/RBP-Jk) reporter is premixed with constitutively expressing Renilla (sea pansy) luciferase vector, which serves as an internal positive control for transfection efficiency._x000D_The pack also includes a non-inducible firefly luciferase vector premixed with constitutively-expressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, but without any additional response elements. The negative control is critical for determining pathway specific effects and background luciferase activity._x000D_Additionally, the pack includes cell culture medium (BPS Medium 1) that has been optimized for use with HEK293 cells*. BPS Medium 1 includes MEM medium, 10% fetal bovine serum, 1% non-essential amino acids, sodium pyruvate, and 1% Pen/Strep. Finally, the pack provides the Dual Luciferase (Firefly-Renilla) Assay System. These luciferase reagents provide highly sensitive, stable detection of firefly luciferase activity and Renilla luciferase activity. The dual luciferase reagents can be used directly in cells in growth medium, and can be detected with any luminometer; automated injectors are not required. _x000D_*Note: the kit may be used with other cell lines than HEK293, but an alternate cell culture medium may be required for optimal cell growth,
Description: A competitive ELISA for quantitative measurement of Human Agouti signaling protein(ASIP) in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.
Description: A competitive ELISA for quantitative measurement of Human Agouti signaling protein(ASIP) in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.
Description: A competitive ELISA for quantitative measurement of Human Agouti signaling protein(ASIP) in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.
We did not find any security issues until 17 days after intravitreal injection RO4929097. Conclusion: Inhibiting Notch signaling may be an effective way to prevent retinal fibrosis. This study is of clinical significance in developing treatments to prevent fibrosis in proliferative vitreoretinopathy, proliferative diabetic retinopathy and wet age-related macular degeneration.
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