1 Cancer Cell, Volume 13 Supplemental Data Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator

Primary screening assay A cell based p53-dependent reporter screen of the ChemBridge DIVERSet collection (consisting of 30,000 compounds) was conducted. T22-RGC-∆Fos-lacZ murine cells expressing β-galactosidase under the control of a p53-dependent promoter were seeded in DMEM-10 % FCS medium into 96 well plates. 80 compounds were assayed in each plate at 10 µM. 12 of the remaining wells were left untreated (DMSO only) in order to evaluate the background β-galactosidase activity. 2 wells in each plate were treated with 5 ng/ml and 2 with 50 ng/ml of the established p53 activator actinomycin D. After 18 hours of treatment, medium was removed, cells were lyzed in 50 µl of 1 x Lysis buffer (Promega cat. no. E3971) for 1 hour at room temperature. 150 µl of developer mix containing 80 µg/ml CPRG (Roche cat no. 884308), 0.53 mM MgCl2 and 24 mM β-mercaptoethanol were then added and optical density readings (570 nm) were made after 22 hours. Normalizing these values to the average value obtained for the reading with 5 ng/ml actinomycin D in each plate allows comparison of readings obtained for compounds assayed in different plates and on different dates