Nutlin 3

Metformin Hydrochloride (purity: 99.98%, Cat.no.: HY17471A) and Nutlin-3 (purity: 98.32%, Cat.no.: HY-50696) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). NH₄Cl (purity: ≥99.5%, Cat.no.: A9434) and Pifithrin-α (purity: ≥95%, Cat.no.: P4359) were obtained from Sigma-Aldrich (St Louis, MO, USA). Chloroquine diphosphate (purity: ≥99%, Cat.no.: 4109) was purchased from Tocris Bioscience (Minneapolis, MN, USA). The antibodies against liver Arginase (ab91279), Ornithine Decarboxylase/ODC (ab97395), and Ornithine Carbamoyl-transferase/OTC [EPR19725] (ab203859) were obtained from Abcam (Cambridge, CB20AX, Cambridge, UK).
Antibodies against p53 (# 2524S), AMPKα (#2532S), and β-actin (8H10D10) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phospho-AMPK alpha (Thr172) (AF3423) and phospho-p53 (Ser15) (AF3075) were purchased from Affinity Biosciences (Cincinnati, OH, USA). The Cell Counting Kit-8 was purchased from Dojindo (Mashikimachi, Kumamoto, Japan). Antibodies against p21(AP021) and LC3B(AL221) and the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

The following antibodies were used: WIP1 (sc-130655), p53 (sc-6243), TFIIH (sc-293), importin (sc-137016), p21 (sc-397) from Santa Cruz; pSer15-p53 (#9284), γH2AX (#9718), p38 MAPK Thr180/Tyr182 (#9216S) and p38 MAPK (#9212) from Cell Signaling Technology); γH2AX (05-636, Millipore); MDM2 (Calbiochem); Alexa Fluor-labelled secondary antibodies (Life Technologies); anti-BrdU FITC-conjugated antibody (#347583, BD Biosciences) and anti-pSer10-H3 antibody (Upstate). Doxorubicin hydrochloride (Sigma), GSK2830371 and nutlin-3 (both MedChem Express) were diluted in DMSO and used at indicated doses. Resazurin, neocarzinostatin (NCS) and carboxyfluorescein diacetate succinimidyl ester (CFSE) were purchased from Sigma.

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.