*4.1. Cell Culture and AKG*

Human osteosarcoma cell lines Saos-2 (HTB-85TM) and HOS (CRL-1543TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The Saos-2 cells were maintained in McCoy's 5A Modified Medium (Sigma-Aldrich Chemicals, St. Louis, MO, USA) supplemented with an antibiotic/antimycotic solution (a/a; Sigma-Aldrich) and 10% fetal bovine serum (FBS; Sigma-Aldrich) in a 5% CO<sup>2</sup> humidified atmosphere at a temperature of 37 ◦C. The HOS cells were grown in Eagle's Minimum Essential Medium (Sigma-Aldrich) supplemented with 10% FBS and a/a. The cells were maintained in a humidified incubator with 5% CO<sup>2</sup> in air at 37 ◦C.

Alpha-ketoglutarate disodium salt dihydrate (Na2AKG × 2H2O; (Sigma-Aldrich) was used in the experiments. Before each experiment, the stock solution of AKG (1 M) was prepared by dissolving the compound in the culture medium. The stock solution was filtered through a sterile syringe filter Millex-GV (Merck Millipore Corporation, Burlington, MA, USA) and diluted in an appropriate culture medium to obtain the required concentrations.

#### *4.2. Cell Proliferation Assays*

The influence of AKG on the proliferation of OS cells was estimated with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution) assay as described previously [71]. Briefly, 4 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well were seeded into 96-well plates in growth medium. After 24 h, the culture medium was removed and the cells were exposed to the dilutions of AKG (2.5–200 mM) prepared in the growth medium with 10% FBS. Following 96-h exposure, the cells were incubated for 3 h with an MTT (Sigma-Aldrich) solution (5 mg⁄mL) and then formazan crystals were solubilized overnight by adding SDS buffer (10% SDS in 0.01 N HCl). The absorbance was determined at a wavelength of 570 nm using an EL800 Microplate Reader (BioTek Instruments, Winooski, VT, USA).

The anti-proliferative activity of AKG was also assessed with the BrdU assay, in which DNA synthesis in proliferating cells was determined after 48 h by measuring bromodeoxyuridine incorporation using a commercial Cell Proliferation ELISA BrdU kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions.

The absorbance of the control wells was taken as 100% and the results were expressed as a percentage of the control. IC<sup>50</sup> values were defined as drug concentrations necessary to inhibit 50% of growth, compared to untreated control cells. IC<sup>50</sup> was obtained using the non-linear regression program GraphPad Prism v5 software (GraphPad Software Inc., San Diego, CA, USA).

### *4.3. Cell Cycle Analysis*

The Saos-2 or HOS cells were seeded into 6-well plates in medium containing 10% FBS without or with AKG (10, 25, or 50 mM). After 48-h treatment, the cell cycle analysis consisting of determination of DNA contents on the basis of PI staining was performed using a flow cytometer (BD FACSCalibur, BD Biosciences, San Jose, CA, USA) and the Cell Quest Pro Version 6.0. for the Macintosh operating system as described previously [72]. Briefly, the cells were washed with PBS and fixed overnight with 80% ethanol at −20 ◦C. Next, the cells were washed with PBS and stained with PI using PI/RNase Staining Buffer (BD Biosciences, BD Pharmingen™, San Jose, CA, USA) for 30 min in darkness at RT. Next, the stained cells were analyzed using FACS Calibur. In total, 10,000 events were measured per sample. The data were analyzed to determine the percentage of cells at each phase of the cell cycle (G1, S, and G2/M).

#### *4.4. Flow Cytometry*

The quantitative analysis of AKG-induced cell death was performed using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (BD Biosciences, BD Pharmingen™, San Jose, CA, USA) and the flow cytometry method as described previously [73]. Briefly, the Saos-2 or HOS cells were seeded into 6-well plates at a density of 7 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well. The next day, the growth medium was replaced with a fresh one containing 2% of FBS supplemented with AKG (5, 10, 25, or 50 mM). In some experiments, the Saos-2 cells were exposed to AKG (50 mM), SP600125 (a selective inhibitor of JNK, 5 µM, Sigma-Aldrich), or a combination of these two compounds. After 72-h incubation, the samples were harvested, washed with PBS, and resuspended in 1 × binding buffer. The cells (1 <sup>×</sup> <sup>10</sup><sup>5</sup> ) were then stained with 5 mM of FITC-Annexin V and 5 mM of PI. After 15-min incubation in the dark at room temperature, the cells were immediately analyzed using a flow cytometer (BD FACSCalibur) with CellQuest Pro Version 6.0 software. All experiments were performed in triplicate and yielded similar results.

The fluorescence-activated cell sorting (FACS) technique was also employed to determine the active form of caspase-3, caspase-9, and caspase-8 in the AKG-treated or untreated OS cells. After 72-h exposure to AKG, a phycoerythrin (PE) Active Caspase-3 Apoptosis Kit (BD Biosciences, San Jose, CA, USA)) and a fluorescein CaspaTag Caspase 9 In Situ Assay kit (Sigma-Aldrich) or a CaspaTag Caspase 8 In Situ Assay kit (Sigma-Aldrich) were used according to the manufacturer's instructions.
