*4.7. PathScan ELISA Assays*

The quantification of the intracellular levels of total and phosphorylated JNK, ERK1/2, p38, and AKT kinases and the contents of cyclin D1 and p21 proteins in the treated cells was carried out using the PathScan® ELISA kits: Total SAPK/JNK Sandwich ELISA Kit, Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit, Total p44/42 MAPK (Erk1/2) Sandwich ELISA Kit, Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit, Phospho-p38 MAPK (Thr180/Tyr182) Sandwich ELISA Kit, Total Cyclin D1 Sandwich ELISA Kit, Total p21Waf1/Cip1 Sandwich ELISA Kit (Cell Signaling Technology Danvers, MA, USA), and p38 MAPK alpha ELISA Kit (Abcam, Cambridge, UK) according to the manufacturer's instructions as described previously [36]. Briefly, the Saos-2 cells (1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL) were incubated in culture medium (2% FBS) without or with the selected concentrations of AKG (10, 25, and 50 mM) in 10-cm diameter plastic plates. In some experiments, the cells were pre-treated with SP600125 (a selective inhibitor of JNK1/2, Sigma-Aldrich) at a concentration of 5 µM for 1 h. After 6, 24, or 48 h of incubation, the media were removed and the cells were rinsed with ice-cold PBS

(Sigma-Aldrich). Then, the cells were lysed in lysis buffer (included in the kits) supplemented with PMSF (Sigma-Aldrich) and protease and phosphatase inhibitor cocktail (Sigma-Aldrich) according to the manufacturer's protocol. Total cell lysates were centrifuged at 14,000× *g* rpm, 5 min at 4 ◦C, and kept at −80 ◦C until analysis. Before the assay, the total protein concentrations in the cell lysates were determined with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and samples containing equal amounts of total proteins per 100 µL of the sample diluent were subjected to ELISA. The optical density was measured using an E-max Microplate Reader (Molecular Devices Corporation, Menlo Park, CA, USA).
