pcmv rorγ plasmid  (Novus Biologicals)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor

doi: 10.1073/pnas.1707965114

Figure Lengend Snippet: OCDO causes GR nuclear localization, regulates GR transcriptional activity and induces cell cycle progression. (A) Representative immunofluorescence image of three different experiments of GR nuclear localization. MDA-MB231 cells were cultured in complete medium with dextran-coated charcoal-stripped FBS for 24 h and then treated for 24 h with either the solvent vehicle (control), 0.5 µM cortisol, or 5 µM OCDO and analyzed by indirect immunofluorescence with a confocal microscope (LSM 780; Zeiss) confocal microscope, 63×, using anti-GR (1:100) primary rabbit monoclonal antibody and a secondary rabbit Alexa fluor-488 secondary goat polyclonal antibody. The nuclei were stained with DAPI. Cells (100 per slide) were analyzed for nuclear GR localization using Jacop plugin in ImageJ v1.51. (Scale bars, 10 µm.) The experiments were performed in triplicate. (B) qRT-PCR analysis of SGK1 and MKP1 mRNA expression in MDA-MB231 tumor cells treated either with solvent vehicle (control), 0.5 µM cortisol, 5 µM OCDO, or 0.5 µM cortisol + 5 µM OCDO for 24 h. Data are the means ± SEM of three experiments performed in triplicate, **P < 0.01, ***P < 0.001, one-way ANOVA, Tukey’s posttest. (C) Effect of OCDO on the transcriptional modulatory activity of RORα, RORγ, and FXR. (Left) HEK293T cells were cotransfected with pSG5-RORα and a RORE-Luc plasmid and treated with or without 10 µM 7-ketocholestrol (7KC) or 1 or 10 µM OCDO for 24 h. (Center) Cells were transfected with pCMV-RORγ and a RORE-Luc plasmid and treated with the solvent-vehicle, 1 or 10 µM OCDO with or without 10 µM SR1078 for 24 h. (Right) Cells were transfected with pSG5-FXR, pSG5-RXR, and the tk-EcRE-Luc plasmid and incubated with either the solvent-vehicle, 1 or 10 µM OCDO with or without 10 µM chenodeoxycholic acid (CDCA). Luciferase activity was measured and normalized per microgram of proteins then expressed as percent activity relative to vehicle-treated cells and represented the mean of three independent experiments performed in triplicate (±SEM), ***P < 0.001, one-way ANOVA, Tukey’s posttest. ns, not significant. (D) Representative cell cycle analysis by flow cytometry of three independent experiments. ShC and shGR6 MDA-MB231 cells cultured for 48 h in serum-free medium were treated with the solvent vehicle (control) or 5 µM OCDO for 7 h. Cells were stained with BrdU and propidium iodide. The percentages of cells within the G0/G1, S, and G2/M phases of the cycle were calculated using FlowJo software. Numbers in the panels indicate the percentages of cells within G0/G1, S, and G2/M phases of the cell cycle.

Article Snippet: The NEON Transfection system was purchased from Invitrogen, the BrdU cell proliferation Elisa kit was from Roche Diagnostics or Sigma, and the following plasmids were from OriGene: 11HSD1: sc109325; 11HSD2: sc122552 or RG207796; H6PD: sc117481; DHCR7: sc110871; and D8D7I (EBP): sc116006. pCMV-RORγ plasmid (NBP2-25278) was from Novus biologicals; pSG5-hGR was a gift from H. Richard-Foy, CNRS, Toulouse, France; pSG5-hRORα was a gift from V. Laudet, CNRS, Banyuls-sur-Mer, France; RORE-Luc was from B. Staels, the Pasteur Institute, Lille, France; pSG5-hRXRγ was a gift from M. Oulad-Abdelghani, INSERM, Strasbourg, France; and pSG5-hFXR and tk-EcRE-Luc were gifts from R. M. Evans, The Salk Institute, La Jolla, CA.

Techniques: Activity Assay, Immunofluorescence, Cell Culture, Microscopy, Staining, Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Incubation, Luciferase, Cell Cycle Assay, Flow Cytometry, Software