p53 transcriptional activation (CPRG) cell reporter assay

ARN8 cells were seeded in a 96-well plate at a density of 20,000 cells/well in a volume of 100 μl of fully supplemented growth medium. Following a 24-h incubation, the cells were treated with 100 μl of the corresponding compound titration diluted in growth medium either nonsupplemented or supplemented with 100 μm uridine, 1 mm DHO, or 1 mm OA. After 18 h of treatment, the medium was removed, and the cells were washed once with 1× PBS (Hyclone). Subsequently, 50 μl of 1× reporter lysis buffer (Promega #E4030) was added to each well, and the plates were stored at –20 °C for at least 2 h. After thawing the cell lysate, 150 μl of CPRG mix consisting of 0.1 m phosphate buffer, pH 7.5 (0.2 m Na₂HPO₄, 0.2 m NaH₂PO₄, distilled H₂O), 4 mg ml⁻¹ chlorophenol red-β-d-galactopyranoside monosodium salt (Roche Applied Science #884308) diluted in 0.1 m phosphate buffer, pH 7.5, 0.1 m MgCl, 4.5 m β-mercaptoethanol (Sigma–Aldrich #M6250) was added to each well. β-Gal activity was measured after 24 h at 590 nm on a spectrophotometer.