PCR with 50 mC of redivue dCTP32 (Amersham), using specific primers for mouse PPP1CA. The purified probe was denatured and added to the hybridization solution. The hybridization was performed overnight at 65uC. After extensive whashing, the membrane was exposed to a Biomax MS film (Kodak). Data Analysis The data consisted of the expression values of 122 transcrip- tional targets of p53 in different cellular conditions, which led either to proliferation or to growth arrest. Clustering analysis. Hierarchical clustering was performed using the function hcluster (package amap) of the free statistical software R (Ihaka and Gentleman, 1996). Before statistical analysis, gene expression levels were standardized gene by gene across all conditions using the median and interquartile range (IQR). The cellular conditions were clustered using Ward linkage and uncentered Pearson metric tests. The results were visualized and analyzed with TreeView (M. Eisen; http://www.microarrays. org/software). The expression level of each gene, relative to its median expression level across all conditions, was represented by a color, with red representing expression greater than the median, green representing expression less than the median, and color intensity representing the magnitude of the deviation from the median. Feature selection. The problem of extracting a robust set of predictors for the proliferating status of the different cellular conditions has been formulated as a least squares regression problem. Since the number of genes is much larger than the number of conditions, we used penalized regression methods. The standard penalty used in so-called ridge regression is given by the L2-norm of the vector containing the regression coefficients. Such penalty allows stabilizing the ordinary least squares estimate, but typically will retain all regression coefficients so that no selection of the relevant variables (genes) may be done. To perform the selection task, we used an L1-norm penalty, as is done in lasso regression. This type of penalty is known indeed to promote sparsity, i.e., to force many regression coefficients to be zero; this obviates the need for pre-selection of the data. However, a known drawback of the L1 penalty for variable selection is that in a group of highly correlated genes, it may pick up only one representative. We therefore also used combined L1- and L2-norm penalties to select sparse groups of highly correlated genes; this is done in the so-called ‘‘elastic net’’ proposed in Zou and Hastie, 2005, [46]. To compute the corresponding penalized least-squares solutions, we applied the iterative thresholding algorithm developed in Daubechies et al, 2004. [47], which is simple to implement, robust to measurement errors and works well for high-dimensional data. Despite the small number of conditions, some standard validation tests (such as leave-one-out, label and gene permutation, bootstrap sampling) were performed. Transcriptional Assays For transient transfection of cells, we seeded 2–46105 H1299 cells per well in six-well plates. After 24 h, transfections were performed by the calcium chloride method and JetPEI reagent (Polytransfection, Illkirch, France) according to the manufacturer’s recommendations. For both transfection methods, we used 1.5– 2 mg each of the reporter plasmids pGL3-13X, pGL3-Bax and pGL3-p21 in the presence or absence of pBABE puro p53 wt (0.6– 0.75 mg) and pLSXN Ras val 12, or active mutants of the PI3K or Raf pathway (0.6–0.75 mg). Renilla luciferase plasmid was used as an internal control for transfection efficiency. The total amount of DNA within the p53 Response to Senescence experiments was kept constant by adding empty vector plasmid DNA to the transfection mixtures. Reporter gene assays were performed with the Dual-LuciferaseH Reporter Assay System (Promega, USA) 48 h after transfection and the results were measured with a Victor2V luminometer. The activity of the reporter luciferase was expressed relative to the activity in renilla vector-transfected cells. Similar results were obtained in at least three different experiments. All results were compared to the control and are shown in the figures as the mean6S.D. of independent triplicate cultures. Western Blot Cells were prepared in lysis-buffer and proteins were separated on SDS-PAGE gels, transferred onto PVDF membranes (Im- mobilon-P, Millipore) and immunostained. The following primary antibody was used: anti-p53FL393 (Santa Cruz 6243, diluted 1:1000), anti-PP1a (from Calbiochem) anti-Rb: G3-245 from BD Pharmingen; and horseradish peroxidase-labeled rabbit anti- mouse (Promega diluted 1:5000) and goat anti-rabbit (Calbiochem 401315, diluted 1:4000) secondary antibodies. Proteins were visualized using the ECL detection system (Amersham Biosciences, Buckinghamshire, UK). Immunofluorescence Immunostaining and confocal analysis for 53BP1 and cH2AX foci. Cells were seeded onto glass cover slips and cultured for 8 h at 39uC. Then we placed the cells at 39uC and 32uC. After 24 h (cells at 39uC and 32uC) and 48 h, 96 h and 144 h (cells at 32uC), cover slips were fixed in 4% paraformaldehyde for 5 min at room temperature, washed twice with PBS, permeabilized in Triton X-100 0.5% in PBS for 5 min and washed twice more with PBS. Samples were incubated in blocking solution (PBS containing 3% bovine serum albumin) at 37uC for 15 min, followed by incubation for 30 min at 37uC with anti-phospho-Histone H2A.X (Ser139) antibody (Millipore 05- 636) or anti-53BP1 antibody (Novus Biologicals NB100-304) diluted 1:100. After washing with PBS, cells were incubated with species-specific Alexa 488-conjugated secondary antibody diluted 1:100 in blocking buffer for 30 min at 37uC in the dark. The nuclei were stained with Hoechst 33258 diluted 1:1000 for 3 min at room temperature in the dark prior to mounting with mowiol (Calbiochem). Images were collected by confocal laser microscopy (model TCS-SP2-AOBS, Leica, Germany). Immunostaining and confocal analysis for PPP1CA and pRb co-localization. Cells were seeded onto glass cover slips and cultured for 8 h at 39uC. Then we placed the cells at 39uC and 32uC. After 24 h (cells at 39uC and 32uC) and 48 h (cells at 32uC), cover slips were fixed in 4% paraformaldehyde for 5 min at room temperature, washed 2 times with PBS, permeabilized in Triton X-100 0.5% in PBS for 5 min and washed again 2 times with PBS. Samples were incubated in blocking solution (PBS containing 3% bovine serum albumin) at 37uC for 15 min, followed by incubation for 30 min at 37uC with anti-human Retinoblastoma Protein (RB) monoclonal antibody (BD Pharmingen 554136) diluted 1:100. After washing with PBS, cells were incubated with species-specific Alexa 488-conjugated secondary antibody diluted 1:100 in blocking buffer for 30 min at 37uC in the dark. Then, cells were incubated with anti-Protein Phosphatase 1a, C-terminal antibody (Calbiochem 539517) diluted 1:100. After washing with PBS, cells were incubated with species-specific Alexa 633-conjugated secondary antibody diluted 1:100 in blocking buffer for 30 min at 37uC in the dark. The nuclei were stained with Hoechst 33258 diluted 1:1000 for 3 min at room temperature in the dark prior to mounting with mowiol PLoS ONE | www.plosone.org 12 September 2008 | Volume 3 | Issue 9 | e3230