Figure 2. Colony formation ARNTL, RBCK1 and TNIP1 shRNA vectors. Cells were infected with shRNA vectors targeting ARNTL, RBCK1 and TNIP1 and control shRNA vectors targeting GFP, p53, 53BP1 and p21. Cells were infected at 32uC and shifted to 39uC 2 days after infection. After three weeks culture at 39uC, the cells were fixed and stained. doi:10.1371/journal.pone.0004798.g002 Knockdown of target genes by shRNA vectors Next, we investigated if the shRNA vectors targeting ARNTL, TNIP1 and RBCK1 also reduced mRNA levels of their cognate target genes. BJtsLT cells were infected at 32uC and shifted to 39uC 3 days after infection. When colonies appeared, RNA was isolated and subjected to quantification by QRT-PCR. The result from the QRT-PCR showed that enriched shRNA vectors targeting ARNTL, RBCK1 and TNIP1 were more potent in decreasing target mRNA than the shRNA vectors that were not enriched (Figure 3a, 3b, 3c). In addition, we tested for both ARNTL and TNIP1 if protein levels were also affected by the shRNA vectors. For ARNTL we co-expressed the three shRNA vectors together with a cDNA encoding hARNTL in Phoenix cells. From the western blot analysis it was clear that only the vectors that could produce colonies also induced potent knock- down of protein expression, thus linking gene knockdown to the p53 growth arrest bypass phenotype (Figure 3d). Knockdown of TNIP1 was determined by analyzing endogenous protein levels in BJtsLT cells (Figure 3e). As can be seen only the vectors that are enriched in the barcode screen and validated to enable colony growth at 39uC were able to reduce endogenous TNIP1 protein levels. We conclude that by limiting the hit selection to genes that are targeted by two independent shRNAs we have only selected ‘on-target’ hits from a complex library. Knockdown of ARNTL, RBCK1 and TNIP1 in BJtsLT cells leads to reduced p21CIP1 levels p21CIP1 is one of the critical effectors of p53 to induce a cell cycle arrest [23]. This is further supported by our identification of two shRNAs targeting p21CIP1 in the list of outliers of the BJtsLT screen. We therefore examined the effect of knockdown of ARNTL, shRNA Screen Modulators of p53 RBCK1 and TNIP1 on p21CIP1 induction. We tested the effects on p21CIP1 in the BJtsLT system that we used for the initial screen. When BJtsLT cells are shifted to 39uC, a rapid increase in p21CIP1 protein levels is observed (Figure 4a). As expected, this p21CIP1 induction is attenuated in cells infected with shRNA vectors targeting p53, p21CIP1 or 53BP1 (Figure 4a). When we used shRNA vectors targeting ARNTL, RBCK1 and TNIP1 we observed a decrease in p21CIP1 levels for those shRNA vectors that produced colonies at 39uC, but not for shRNA vectors that failed to produce colonies (Figure 4a–c). To be sure that the decrease in p21CIP1 protein levels were caused by decreased p21CIP1transcription we also measured p21CIP1 mRNA levels by QRT-PCR (Figure 4d). All shRNAs that could produce colonies at 39uC also showed a decrease in p21CIP1 mRNA. This result suggests that the knockdown of ARNTL, RBCK1 and TNIP1 leads to a decreased transcriptional activity of p53 towards its target p21CIP1. Recently multiple reports have discussed the relationship between cancer and circadian rhythm [24–26]. ARNTL is a core component of circadian rhythm transcriptional machinery [20,27]. ARNTL binds to CLOCK and together they regulate expression of 1,000s of genes in a circadian timing [28,29]. Genes regulated in a circadian fashion are involved in cell cycle, detoxification and other processes [30]. Therefore we decided to test if ARNTL is involved in the regulation of p21CIP1 expression in other cell systems. Reduced p21CIP1activation after DNA damage in HCT116 cells with ARNTL knockdown Normal human cells arrest either in G1 or S phase of the cell cycle after encountering DNA damage to repair the DNA, thereby preventing accumulation of mutations in the genome of daughter cells. The G1 phase cell cycle arrest is p53 dependent and mainly executed by the CDK inhibitor p21CIP1 [31,32] . To investigate if ARNTL is also required for the p21CIP1 activation after DNA damage, we infected U2OS osteosarcoma derived cells with different shRNAs targeting ARNTL. The cells were incubated to allow knockdown to take affect, after which cells were irradiated to inflict DNA damage and monitored for p21CIP1 activation. When we compared cells that were infected with a shRNA vector targeting p53 to cells infected with a control shRNA vector, we observed lower p53 and p21CIP1levels after c-radiation. In the cells infected with ARNTL knockdown vectors we also observed lower p21CIP1protein levels, but p53 protein levels were unaffected (Figure 5a). This observation suggests that ARNTL can modulate the activity of p53 towards its target p21CIP1. However, we cannot distinguish between a specific effect of ARNTL on p21CIP1and a more general effect of ARNTL on p53 transcriptional activity. ARNTL knockdown also allows bypass a p19ARF induced growth arrest As the BJtsLT cells are quite artificial due to the presence of the SV40 T viral oncogene, we also investigated if ARNTL knockdown could bypass a more physiological p53-induced cell cycle arrest. To address this, we used cells in which we can activate p53 by over-expression of p19ARF. p19ARF inhibits MDM2 function thereby leading to an increase of p53 protein and activation of target genes [33]. Activation of p19ARF leads to a stable p53- dependent cell cycle arrest [34]. To test if ARNTL knockdown can also rescue a p19ARF-induced cell cycle arrest, we infected U2OS cells with the shRNAs targeting ARNTL. After knockdown had taken effect the cells were super-infected with a p19ARF encoding retrovirus. We observed that cells with knockdown of p53 or PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4798