shRNA Screen Modulators of p53 Figure 1. shRNA barcode screen identifies mediators of the p53 dependent cell cycle arrest. a) Schematic outline of the BJtsLT genetic screen. BJtsLT cells were infected with the NKI shRNA library and were either left at 32uC or shifted to 39uC. After 7 days the cells at 32uC had reached confluency and were harvested. Cells at 39uC were harvested after 23 days after which they had formed visible colonies. b) Analysis of the relative abundance of shRNAs recovered from the BJtsLT barcode experiment. Data are normalized and plotted as M, the 2log (ratio Cy5/Cy3), versus A (2log(!intensity Cy36Cy5)). The data are the average of two independent hybridization experiments performed in duplicate with reversed colour. A red box is drawn around the top 100 enriched shRNAs at 39uC. c) Schematic overview of selection criteria used to select hits from the shRNA barcode screen for further validation. doi:10.1371/journal.pone.0004798.g001 However, for 37 of the 42 enriched shRNA vectors, we could clearly demonstrate that they allowed the cells to proliferate at 39uC. Among the genes targeted by these 37 shRNA vectors were all known p53 pathway components. In addition, we also observed rescue of growth arrest by all three genes that were targeted by two individual shRNAs: ARNTL, RBCK1 and TNIP1 (Figure 2). Therefore we decided to focus on these three newly identified genes for which we identified two independent shRNAs. To show the sensitivity and selectivity of the shRNA barcode technique, we decided to test all three shRNA vectors targeting ARNTL, RBCK1 and TNIP1 that are present in the library. As mentioned before, for these three genes we found only two of the three shRNAs to be enriched in the screen, whereas one shRNA vector was not enriched. When we infected all three shRNA vectors independently into BJtsLT cells we found that only shRNAs enriched in the shRNA barcode screen gave rise to colonies (Figure 2). PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4798