isre lucia luciferase reporter gene  (InvivoGen)

Journal: Arthritis Research & Therapy

Article Title: Potential role of RhoA GTPase regulation in type interferon signaling in systemic lupus erythematosus

doi: 10.1186/s13075-024-03263-3

Figure Lengend Snippet: RhoA regulates the type I IFN-stimulated response element (ISRE) and downstream gene expression. A ISRE activity of HEK293T cells transfected with ISRE-luciferase and a Renilla reporter plasmid together with a negative control (Ctrl) or RhoA-targeted siRNA (each at 200 nM). B ISRE activity of HEK293T cells transfected with RhoA-overexpression or control plasmids (each at 100 ng). At 24 h after transfection, the cells were left unstimulated (0 h) or stimulated with IFN-a (1000 U/mL, 6 h). Data in A and B are expressed as fold-change based on relative luciferase activities (ratio of firefly luciferase to Renilla luciferase). C – E The relative expression of OAS1, IFIT3, and CXCL10 mRNAs were determined by quantitative PCR. F The CXCL10 levels in culture supernatants were determined by enzyme-linked immunosorbent assay. Data were assessed in HEK293T ( C , D ) and THP1 cells ( E , F ) at 24 h after transfection of the negative control (Ctrl) or siRNA (200 nM) stimulated cells with IFN-a (1000 U/mL) for 6 h. Bars show the mean ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student’s t- test

Article Snippet: HEK-293 T cells were cultured in a 96-well plate and co-transfected with siRhoA (200 nM) or RhoA expression plasmids (4 mg/mL), or their controls together with a mixture of Renilla vector (10 ng, Promega) and the ISRE-luciferase reporter gene vector (100 ng, Clontech) for 24 h. IFN-a (1000 U/mL) then was added for an additional 6 h of incubation.

Techniques: Expressing, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Negative Control, Over Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Potential role of RhoA GTPase regulation in type interferon signaling in systemic lupus erythematosus

doi: 10.1186/s13075-024-03263-3

Figure Lengend Snippet: The RhoA/Rock Inhibitor Y27632 downregulates type I IFN signaling. A HEK293T cells were co-transfected with ISRE-luciferase and Renilla reporter plasmids for 24 h, then cultured with or without Y27632 (60 μM, 45 min) before the addition of IFN-a (1000 U/mL, 6 h). Luciferase activity was measured by dual luciferase assay. B PBMCs from healthy controls were cultured in the presence or absence of Y27632 (60 μM, 45 min) and stimulated with IFN-a (1000 U/mL) for different times. Western blots show whole-cell lysates harvested at the indicated times. C Histograms show the ratios of phosphorylated to total STAT-1 at the indicated times. The ratio of pSTAT-1/STAT-1 in the absence of Y27632 and IFN-a at 0 min was set at 1. D Relative expression of OAS1 and E CXCL10 mRNA in PBMCs cultured with Y27632 (0, 30, 60, and 90 μM, for 45 min and stimulated with IFN-a (1000 U/mL) for 6 h. Results are the relative expression levels of OAS1 and CXCL10 mRNA normalized to endogenous GAPDH mRNA levels. F CXCL10 levels in PBMC culture supernatants quantified by ELISA. Bars show the mean ± SEM of 3 individual healthy donors; * p < 0.05, ** p < 0.01, A and C versus vehicle by Student’s t- test

Article Snippet: HEK-293 T cells were cultured in a 96-well plate and co-transfected with siRhoA (200 nM) or RhoA expression plasmids (4 mg/mL), or their controls together with a mixture of Renilla vector (10 ng, Promega) and the ISRE-luciferase reporter gene vector (100 ng, Clontech) for 24 h. IFN-a (1000 U/mL) then was added for an additional 6 h of incubation.

Techniques: Transfection, Luciferase, Cell Culture, Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay