Geiparvarin Inhibits OS Metastasis through Upregulation of ANGPTL4 Expression by Inhibiting miRNA-3912-3p Expression
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Background. Geiparvarin (GN) is a natural compound with anticancer activity. However, the effect of GN on osteosarcoma (OS) and the anticancer mechanism of GN are still unclear. Methods. Cell viability was measured by MTT assay. Invasion and migration were measured by transwell assay. The miRNAs, genes, and signaling pathways affected by GN were confirmed by whole-genome sequencing and bioinformatics analysis. The expression level of mRNA and protein was measured by qRT-PCR and western blot. Animal experiment was performed for confirming the GN anticancer effect and side effect in vivo. Results. Our results show that GN significantly inhibits OS cell growth and metastasis in vitro. In vivo experiment also showed that GN dramatically suppressed OS lung metastasis and no side effects were found. GN treatment inhibited OS metastasis through upregulating the ANGPTL4 expression. In addition, GN inhibited the expression of miR-3912-3p, which targets ANGPTL4. Conclusion. Our data clearly indicate that GN is a candidate drug for OS treatment, and GN plays its role through miR-3912-3p/ANGPTL4 in OS.Keywords:
ANGPTL4
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Background : Cell viability is an important factor in radiaon therapy and thus is a method to quanfy the effect of the therapy. Materials and Methods : The viability of human hepatoma (HepG2) cells exposed to radiaon was evaluated by both the MTT and Trypan blue assays. The cells were seeded on 96 well-plates at a density of 1 x 10 4 cells/well, incubated overnight, and irradiated with 1-100 Gy. Results: The cell viability was decreased in a dose- andme- dependent manner when using the Trypan blue assay, but no significant changes in the response to dose could be detected using the MTT assay. It indicated that the MTT assay was not efficient at a cell density of 1 x 10 4 cells/well on 96 well-plates to determine cell viability. Subsequently, the relaonship between cell viability and lower cell density (1 x 10 3 , 3 x 10 3 , and 5 x 10 3 cells/well) was invesgated. A cell density of 1 x 10 3 was found to be the most effecve when using the MTT assay. Results show that the cell density is most important when using the MTT assay in 96 well-plates to follow in radiaon effects. Furthermore, the radiao n-induced cell viability dependent on cell density was confirmed by using the tradional Clonogenic assay. Conclusion: Our results suggest that the MTT and Trypan blue assays are rapid methods to detect radiaon-induced cell viability of HepG2 cells in about 3 days as compared with 14 days of assayme in the Clonogenic assay. To obtain accurate cell viability measures using both rapid assays, an incubaonme of at least 3 days is needed a6er irr adiaon.
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Viability assay
Clonogenic assay
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Propidium iodide
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To evaluate the expression of uncoupling protein 2 (UCP2) in a retinal pigment epithelium cell line (ARPE-19), under oxidative stress (OS).ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2O2; 0, 150, 300, 500, 700, and 900 µmol/L) for 24h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H2O2 was investigated by reverse transcription-polymerase chain reaction (RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry (FCM). Further, UCP2-siRNA treated cultures were exposed to H2O2 (0, 75, 150, and 300 µmol/L) for 2h and cell viability determined by MTT assay.Cells treated with higher concentrations of H2O2 appeared shrunken; their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H2O2. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 µmol/L H2O2 (P<0.05). Cell metabolic activity was decreased with increased concentrations of H2O2 as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H2O2 alone (P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H2O2-treated groups compared with controls (P<0.05). FCM analysis showed that cell reactive oxygen species (ROS) levels were increased in H2O2-treated groups and further upregulated by UCP2-siRNA treatment (P<0.05).Expression levels of UCP2 are decreased in ARPE-19 cells treated with H2O2. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H2O2 alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.
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Chlorogenic Acid
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Objective To assess the role of MTT colorimetric assay in measuring mitochondrial succinate-dehydrogenase(SDH)and establish a new way to determine pretransplantational cell viability.Methods Human or rat testicular Leydig cell suspensions were incubated with MTT medium,isopropanol was used to dissolve MTT fornlazan,then the absorbance of supertants was measured at 563 nm wavelength.Cell culture and testosterone production were used to aaseas the reliability of MTT colorimetric assay with conlparimn with Trypan bluedye.Results 1 g/L MTT concentration,3 h incubation,and impropanol as a solvent were the best optimum for the cell culture in vitro and testosterone determination showed that MTT colorimetric assay was more sensitive and accurate than Trypan bluedye.MTT colorimetric assay was employed to successfully measure cell viability for 10 cases of human testicular Leydig cell transplantation.Conelusion MTT colorimetric assay provided a reliable,objective,accurate method for determining the pretransplantational cell viability.
Key words:
MTT colorimetric assay; Cell/viability; Cell/transplantation; Trypan btuedye
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Abstract Metabolic viability based high throughput assays like MTT and MTS are widely used in assessing the cell viability. However, alteration in both mitochondrial content and metabolism can influence the metabolic viability of cells and radiation is a potential mitochondrial biogenesis inducer. Therefore, we tested if MTT assay is a true measure of radiation induced cell death in widely used cell lines. Radiation induced cellular growth inhibition was performed by enumerating cell numbers and metabolic viability using MTT assay at 24 and 48 hours (hrs) after exposure. The extent of radiation induced reduction in cell number was found to be larger than the decrease in MTT reduction in all the cell lines tested. We demonstrated that radiation induces PGC-1α and TFAM to stimulate mitochondrial biogenesis leading to increased levels of SDH-A and enhanced metabolic viability. Radiation induced disturbance in calcium (Ca 2+ ) homeostasis also plays a crucial role by making the mitochondria hyperactive. These findings suggest that radiation induces mitochondrial biogenesis and hyperactivation leading to increased metabolic viability and MTT reduction. Therefore, conclusions drawn on radiation induced growth inhibition based on metabolic viability assays are likely to be erroneous as it may not correlate with growth inhibition and/or loss of clonogenic survival.
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Amyloid beta
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Amyloid (mycology)
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The purpose of this experiment is to investigate the effects of forskolin-mediated cAMP activation on the viability of LPS-treated Schwann cells. The immortalized rat RT4-D6P2T (ATCC #CRL-2768) and S16 (ATCC #CRL-2941) cell lines were cultured and received one of the following treatments: 0.1, 1, or 10 μg/mL of LPS, in N2 media (control) or N2 media supplemented with 2 µM of forskolin, for 1, 3, 12, or 24 hours, and the CyQUANT MTT Cell Viability Assay Kit (Thermo Fisher) was used to perform the viability assay.
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