File S1 - Impact of Glucocorticoid Receptor Density on Ligand-Independent Dimerization, Cooperative Ligand-Binding and Basal Priming of Transactivation: A Cell Culture Model

Figure S1. Whole-cell saturation binding, immunoblotting and fluorescent intensity used to monitor and determine GR levels. (A) COS-1 cells were transfected with GRwt or GRdim (low, medium or high levels) during assays. Immunoblotting was performed (see Material and Methods) on cell lysates and pixels from densitometric analysis of the immunoblots was correlated to GR levels (cpm/mg protein) determined by whole cell saturation binding (see Materials and Methods). A standard curve correlating GR concentrations in cpm/mg protein derived from saturation binding to their respective densitometric values (pixels) from immunoblotting was produced (R2 = 0.9719). This curve was used to monitor and determine GR levels throughout. (B) For FRET assays the relative CFP-GR (F-don) expression levels in individual cells within low, medium and high GR concentration populations were measured and used to monitor GR levels. Exposure times of 1500 ms at 100% light intensity were used. F-don values reflect the CFP signal after 30 minutes of DEX stimulation measured in a region of interest in the nucleus of each individual cell. Cells with an F-don emission of 0–600 where selected for the low [GR] concentration (*, n = 10), F-don signals between 600–1200 for the medium [GR] population (†, n = 7) and F-don of >1200 for the high [GR] population (§, n = 7).Figure S2. Un-induced transactivation increases and fold-induction decrease at higher GRwt concentration through single GRE. Cells were transfected with GRwt or GRdim (low or medium levels) and 3000 ng pΔODLO, a promoter-reporter containing a single GRE. Cells were induced with ethanol, 10−6 M DEX, F, MPA or RU486 for 24 hours. Luciferase activity was determined and relative light units (RLU) were normalized against protein concentrations. (A) Un-induced RLU/mg protein values following 24 hours ethanol stimulation. Statistical analysis was through two tailed unpaired t tests of low GRwt concentration against medium GRwt concentration (†††P§§§PB) Maximal induction and (C) fold-induction (calculated as maximal induction normalized to un-induced induction) were plotted. Statistical analysis was through one-way ANOVA followed by Dunnett's post-test comparing un-induced (ethanol) conditions to the ligand-induced conditions within the low (†††P§P§§P50) of transactivation in a range of ligands. Cells were transfected with GRwt or GRdim (low or medium levels) and pTAT-GRE2-Elb-luc. Cells were induced with ethanol or a range (10−12 M to 10−5 M) of F, MPA, or RU486 for 24 h. Luciferase activity was determined and relative light units normalized against protein concentrations. Sigmoidal dose-response curves where fitted to the experimental data which generated the potency (Log EC50), maximal induction (Bmax) and fold-induction. Statistical analysis was carried out on logEC50-values using one-way ANOVA followed by Newman-Keuls post-test: (*P†P††P†††P (DOC)