RNA was reverse transcribed and used to perform qPCR in triplicate with SYBR GREEN I master mix (Atila Biosystem) on ABI PRISM 7900HT (Applied Biosystems) using primer pairs provided in Methods S1. For quantifying gene expression, qPCR was performed using RNA either from compound or mock treated cells in duplicate in two biological repeats. Transcript quantifica- tion was measured by comparison with standard curves as described above. All results were normalized against GAPDH, and coefficient of variation was calculated. Flow cytometry Cell cycle distribution and cell death were assayed with flow cytometry (LSR, Becton Dickinson). Cells were harvested and fixed in 70% ethanol at 220uC followed by RNaseA treatment and staining with propidium iodide. A total of 10,000 counts were collected and data were analyzed using ModFit LT 3.1 software. Cells present in sub-G1 population were regarded as non-viable. In vivo tumor experiment The animal protocol was approved by the Experimental Animal Committee of Provincial Government of Southern Finland (STH401A), and the animal studies were carried out according to the approved guidelines. An orthotopic model of pleural effusion lymphoma was used to assess the anti-tumor activities of BMH-15 and -22. Female 5–7 weeks old NOD-SCID mice (Charles River) were injected intraperitoneally (i.p.) with 16107 pleural effusion lymphoma cells carrying a luciferase reporter (BC3luc cells) [26]. For imaging, D-luciferin (100 mg/kg) (Synchem OHG) was injected i.p. followed by imaging 15 min post-injection using Xenogen In Vivo Imaging System (Caliper Life Sciences). Tumor-bearing mice, as verified by biolumines- cence, were treated by i.p. injection of the compounds starting on day four post-implantation. The mice received 20 mg/kg Novel p53 Pathway Activators compounds BMH-15 and -22 three times a week, while control mice received only DMSO. The mice were imaged prior each treatment. Bioluminescence was quantified and plotted as total flux within given region of interest (ROI) using Igor Pro Carbon analysis software (Caliper Life Sciences). Statistical analysis was carried out using Linear Model ANOVA using R software. P values less than 0.05 were regarded as statistically significant. Statistical analysis Shown are means 6 SD of at least two independent experiments. Statistical analysis was performed by Fisher’s exact t test. Data on animal studies was performed using Linear Model ANOVA using R software. Differences were considered statisti- cally significant at P,0.05. Results High-content imaging screen identifies novel p53 activating lead compounds An improved protocol for high-throughput cell-based screening was devised. This consisted of generation of a cell line expressing red-emitting fluorescent protein under the control of a p53 consensus promoter (p53DsRed) [21]. In contrast to the previously published screens using a similar reporter element, we adapted the screen to a strictly cell-based analysis using a fully automated high- content imaging and quantitative image analysis platform. This facilitated scoring of signal intensities in individual cells allowing detection of the reporter activation under conditions where p53 activation leads to a significant reduction in cell viability (Figure 1A). We first validated the p53 high-content imaging assay and screened a library consisting of 2000 therapeutic and experimental drugs. We identified and further validated eleven drugs that Figure 1. Cell-based high-content imaging screen. A) Screen outline. B) Spectrum Collection experimental drug library was analyzed using the p53 reporter screen. Primary hits were validated at 10-fold concentration range. Color code; red, TOP1/2 inhibitors; blue, DNA intercalating agents; black, other. C) Chemical structures of hit compounds in the large-scale small-molecule compound screen. doi:10.1371/journal.pone.0012996.g001 PLoS ONE | www.plosone.org 3 September 2010 | Volume 5 | Issue 9 | e12996