U-2 OS cells were transfected with the BD Clontech pTET-OFF regulatory plasmid to establish the U-OFF parental cell line. MluI and EcoRV ends were generated onto the coding region of hGRa using PCR amplification of the pCMVhGRa plasmid. The pTRE2hyg vector was digested with MluI and EcoRV and the two DNAs were ligated to form the pTRE2hGRa plasmid (Lu and Cidlowski). Site-directed mutagenesis was then performed to make pTRE2N363S. The wild type hGR and the N363S mutant were individually transfected into the U-OFF cells and clones were selected which stably expressed either hGRa or N363S using 200 mg/ml of geneticin and 500 mg/ml of hygromycin. Several clones were obtained for each receptor, and the receptor levels were compared using western blot analyses. In these cell lines, the expression of hGR can be repressed by the addition of tetracycline or the derivative doxycycline to the media. U-2 OS (human osteosarcoma) cells were maintained in DMEM/F-12 supplemented with 10% FCS:CS, 2 mM glutamine and pen-strep and selected clones were maintained in the same media with the addition of 200 mg/ml Geneticin and 200 mg/ml hygromycin. All cells were maintained in a humidified, 5% CO2 atmosphere. For the Microarray: U-2 OS cells stably expressing either wild type hGR or N363S were treated for six hours with 10 nM dexamethasone or vehicle, and total RNA was isolated using the Qiagen RNeasy midi kit (Qiagen, Valencia, CA).
Data processing
Gene expression analysis was conducted using Agilent Human1Av2 arrays (Agilent Technologies, Palo Alto, CA). Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer?s protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters.
