Nutlin-3 Induced Growth Suppression

The RPE-TP53-KO cells, stably reconstituted with pIRES2-EGFP-p53 WT or its variants, were seeded in 96-well plates (500 cells/well) and cultivated in the presence of nutlin-3 (0.5 μM, MedChem Express) for 7 days. Resazurin (30 μg/mL) was added to the growth media and the resulting fluorescence signal (Ex = 560 nm, Em = 590 nm) was measured after 1 h using the EnVision plate reader (PerkinElmer), as previously described elsewhere^{54 (link)}. The level of cell growth suppression induced by nutlin was normalized to the wild-type p53. Samples were measured in hexaplicates in three independent experiments. The bars indicate standard deviation. Any statistical significance was determined by two-sided t-test using Prism software.