p53 Response to Senescence Figure 7. Oncogenic Ras enhances p53-transcriptional activation. A, B and C) p53(2/2), p53(2/2); MDM2(2/2) or p19(2/2) MEFs were transfected as indicated with plasmids carrying luciferase and the indicated genes. Luciferase activity was measured as indicated in the M & M. A) Assays were performed in p53(2/2) MEFs using different promoters responding to p53 (p21waf1, Bax and synthetic p53x13 carrying only the p53 binding site repeated 13 times). B) Only the Bax promoter was used in p53(2/2) MEFs. C) Only the Bax promoter was used in p53(2/2), double p53(2/2); MDM2(2/2) or p19(2/2) MEFs. Data show the averages of three independent experiments. doi:10.1371/journal.pone.0003230.g007 For infections, the culture medium was replaced by the viral supernatant, and then the culture plates were centrifuged (1 h at 1,500 rpm) and incubated at 37uC for 16 h. The medium was changed and cells were split 24 h later. Infected cell populations were selected in hygromycin (20 mg/ml) for pWZLHygro-based vectors and in blasticidin (2 mg/ml) for pWZLBlast-based vectors. Northern assays Total RNA was isolated from cells using the TRI-REAGENT method (Molecular Research Center, Cincinnati, OH) according to the manufacturers instructions. A reverse transcription was done for each sample (20 mg of total RNA) using MMLV reverse transcriptase (Promega), oligo dT primer and dCTP32-labeling nucleotide. The cDNA 32P-labeled probes were hybridized to the p53 target gene array membrane (TranSignal, Panomics, CA, USA) at 42uC overnight. After removing excess substrate by gently washing twice with 26SSC+0.5% SDS and 0.16SSC+0.5% SDS at 62uC, the membranes were exposed to BioMax Films (Eastman Kodak Company, NY, USA). The assay normalization was done selecting b-actin as the control housekeeping gene. Analysis was done using the GS-800 Calibrated DensitometerH and the Quantity OneH program from Bio-Rad. Each experiment for each condition was performed indepen- dently at least twice, the data quantified and normalized for the value of b-actin (a gene with transcription that is independent of p53). Raw data for all conditions were normalized against an internal control, b-actin, and then compared to normal proliferating MEFs. PPP1CA Northern Blot. Total RNA was extracted using RNAzolB. 10 mg of total RNA were run in formaldehyde-agarose gels and transferred to a Hybond membranes. The membrane was pre-hybridized during 4 hours at 65uC. The probe was labeled by PLoS ONE | www.plosone.org 11 September 2008 | Volume 3 | Issue 9 | e3230