*4.8. Cell Migration Assay*

The wound-healing assay was used to evaluate the influence of AKG on the migration of osteosarcoma cells in vitro. The Saos-2 (3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL) and HOS (2.5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL) cells were seeded into 3-cm diameter plastic plates and cultured until confluence. Afterwards, wounds were made in the monolayers with a sterile 100-µL pipette tip. The cells were washed twice with PBS to remove cell debris, and fresh growth media without or with AKG (5, 10, 25, and 50 mM) were added. The gap width of the wound in one dish (wound control) was measured and recorded immediately after wounding. After 24-h incubation, the gap width of the wounds in the other plates was measured and contrast-phase images were taken using an inverted microscope Olympus CKX41 (Olympus Optical Co, LTD, Tokyo, Japan) and analyzed with Image Processing (CellSans) software. The inhibition of migration (fold change of the control) was calculated as the quotient of the mean width of the gap in the AKG-treated culture and the mean width of the gap in the control culture.

### *4.9. Cell Invasion Assay*

The OS cell invasion was estimated with the CultureCoat® 24 Well Low BME Cell Invasion Assay (Trevigen, Inc., Gaithersburg, MD, USA) according to the manufacturer's instructions. The Saos-2 or HOS cells (both 1.0 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL) suspended in medium with 1% of FBS and supplemented with AKG (5, 10, 25, or 50 mM) or without AKG (control) were seeded into BME-coated inserts (8 µm) in the wells of a 24-well plate. Medium with 10% FBS (as a chemoattractant) was added into the bottom chamber. After 24 h of incubation, the number of OS cells invading the bottom surface of the insert membrane was quantified based on the amount of free Calcein generated from Calcein AM by migrating cells, as indicated in the manufacturer's instructions. The Calcein fluorescence level was measured by means of a Perkin Elmer Victor™ plate reader (PerkinElmer, Waltham, MA, USA)
