renilla luciferase expression plasmid (prl-tk plasmid)  (Promega)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Srebf and Runx3 regulate hepatic HMG-CoA reductase and intracellular cholesterol under hypoxia

doi: 10.1007/s00018-025-05736-3

Figure Lengend Snippet: Effect of hypoxia on SHR/WKY- Hmgcr promoter activity. A Modulation of SHR/WKY- Hmgcr promoter activity upon hypoxia. BRL 3A cells were transfected with SHR/WKY -Hmgcr promoter constructs and subjected to 6 h of hypoxia. The cells were lysed for luciferase and Bradford assays. The promoter activities were normalized to Renilla luciferase, which was used as an internal control. The results are mean ± S.E of triplicate values. One-way ANOVA with Newman-Keuls multiple comparison test was used to determine the statistical significance,* p < 0.05, ** p < 0.01 when compared to WKY, and ## p < 0.01, ### p < 0.001 when compared to corresponding normoxia condition. BRL 3A cells, subjected to hypoxic stress for 6 h, were stained with Filipin III dye (50 µg/ml) to view intracellular cholesterol and observed under fluorescence microscope. B Fluorescence microscopy images of the filipin-stained BRL 3A cells. Scale bar: 100 µm. C Quantification of total corrected fluorescence per cell was carried out after hypoxic stress using ImageJ. D Western blot analysis of total protein isolated from BRL 3A cells after hypoxia treatment. Elevated expression of Hif-1α confirms hypoxia treatment. The decrease in endogenous Hmgcr and Srebf protein levels and increase in Runx3 protein level was also shown in the blot. E Quantification of endogenous Hmgcr, Hif-1α, Srebf and Runx3 protein levels in BRL 3A cells after hypoxia treatment. The normalized protein levels of Hmgcr, Hif-1α, Srebf and Runx3 from three independent blots are shown as a bar plot. F qPCR analysis was performed using gene-specific primers for Hmgcr , Hif-1α , Srebf , Runx3 and the gene expression was normalized to Vinculin . The results are mean ± S.E of triplicate values. Student’s unpaired t-test was used to determine the statistical significance. ** p < 0.01, **** p < 0.0001 with respect to normoxia

Article Snippet: For transient transfections, BRL 3A cells were grown up to 60% confluency in 12-well plates, and co-transfections were carried out in triplicates with 500 ng/well of SHR/WKY- Hmgcr promoter-reporter plasmids and 300 ng/well of Renilla luciferase expression plasmid (pRL-TK plasmid; Promega, USA) using Targefect F1 transfection reagent (Targeting Systems, USA) according to the manufacturer's protocol.

Techniques: Activity Assay, Transfection, Construct, Luciferase, Control, Comparison, Staining, Fluorescence, Microscopy, Western Blot, Isolation, Expressing, Gene Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Srebf and Runx3 regulate hepatic HMG-CoA reductase and intracellular cholesterol under hypoxia

doi: 10.1007/s00018-025-05736-3

Figure Lengend Snippet: Transcriptional regulation of Hmgcr by Hif-1α. A Regulation of SHR/WKY- Hmgcr promoter activity by ectopic expression of HIF-1α shRNA plasmid. BRL 3A cells were co-transfected with 500 ng/well of SHR/WKY- Hmgcr promoter constructs and increasing doses of HIF-1α shRNA plasmid. The promoter activities were normalized to Renilla luciferase. The results are mean ± S.E of triplicate values. One-way ANOVA with Newman-Keuls multiple comparison test was used to determine the statistical significance. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as compared to the basal condition (without a co-transfected HIF-1α shRNA plasmid), # p < 0.05 as compared to the basal WKY- Hmgcr promoter activity. The knock-down of Hif-1α as confirmed by western blot analysis is indicated below the HIF-1α shRNA co-transfection figures. The increase in endogenous Hmgcr and Srebf protein levels and decrease in Runx3 protein level were also shown upon ectopic expression of Hif-1α. Quantification of endogenous B Hmgcr, C Hif-1α, D Runx3 and E Srebf protein levels in BRL 3A cells were done. The Vinculin normalized protein levels of Hmgcr, Hif-1α, Srebf and Runx3 from three independent blots are shown as a bar plot. One-way ANOVA with Newman-Keuls multiple comparison test was used to determine the statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as compared to the basal condition. BRL 3A cells were transfected with HIF-1α shRNA plasmid (1000 ng) and the cells were stained with Filipin III dye (50 µg/ml) and observed under fluorescence microscope to visualize intracellular cholesterol. F Fluorescence microscopy images of the filipin-stained BRL 3A cells. Scale bar: 100 µm. G Quantification of total corrected fluorescence per cell was carried out after hypoxic stress using ImageJ. Student’s unpaired t-test was used to determine the statistical significance. ** p < 0.01 with respect to untreated cells

Article Snippet: For transient transfections, BRL 3A cells were grown up to 60% confluency in 12-well plates, and co-transfections were carried out in triplicates with 500 ng/well of SHR/WKY- Hmgcr promoter-reporter plasmids and 300 ng/well of Renilla luciferase expression plasmid (pRL-TK plasmid; Promega, USA) using Targefect F1 transfection reagent (Targeting Systems, USA) according to the manufacturer's protocol.

Techniques: Activity Assay, Expressing, shRNA, Plasmid Preparation, Transfection, Construct, Luciferase, Comparison, Knockdown, Western Blot, Cotransfection, Staining, Fluorescence, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Srebf and Runx3 regulate hepatic HMG-CoA reductase and intracellular cholesterol under hypoxia

doi: 10.1007/s00018-025-05736-3

Figure Lengend Snippet: Role of Srebf and Runx3 in the regulation of Hmgcr expression under hypoxia. BRL 3A cells were transfected with SHR/WKY- Hmgcr promoter construct in the presence of either A Srebf siRNA (10 nM) or control siRNA (10 nM) followed by hypoxia treatment for 6 h. The promoter activities were normalized to Renilla luciferase. One-way ANOVA with Newman-Keuls multiple comparison test was used to determine the statistical significance. ** p < 0.01 as compared to the control siRNA; ## p < 0.01, ### p < 0.001 as compared to normoxia. To test whether Hmgcr regulation under hypoxia is also influenced by Runx3, the SHR/WKY- Hmgcr promoter activity was assessed upon B knock-down of endogenous Runx3 followed by hypoxia treatment for 6 h. One-way ANOVA with Newman-Keuls multiple comparison test was used to determine the statistical significance. ** p < 0.01 as compared to the control siRNA; ## p < 0.01, ### p < 0.001 as compared to normoxia. The knock-down of Srebf/Runx3 upon co-transfection of Srebf/Runx3 siRNA and increased Hif-1α protein level after hypoxia treatment was confirmed by western blotting analysis. Alteration of Hmgcr protein level upon the knock-down of Srebf/Runx3 was also observed in the western blot represented below the Srebf/Runx3 siRNA co-transfection figures

Article Snippet: For transient transfections, BRL 3A cells were grown up to 60% confluency in 12-well plates, and co-transfections were carried out in triplicates with 500 ng/well of SHR/WKY- Hmgcr promoter-reporter plasmids and 300 ng/well of Renilla luciferase expression plasmid (pRL-TK plasmid; Promega, USA) using Targefect F1 transfection reagent (Targeting Systems, USA) according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Construct, Control, Luciferase, Comparison, Activity Assay, Knockdown, Cotransfection, Western Blot