Assessing Cell Viability and Growth Inhibition

Cell growth and viability were determined using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). SK-MEL-2, SK-MEL-28, and A2058 cells were seeded at 3 × 10³ cells per well in 96-well plates containing 130 μL medium. After overnight incubation, the cells were treated with 0.1% DMSO or PCI-34051 at various concentrations (0.1, 0.3, 1, 3, 10, 30, 100 µM) for 24 h, 48 h, and 72 h. The A2058 HDAC8 KO and overexpression (OE) cells were seeded at 3 × 10³ cells per well in 96-well plates containing 130 μL medium and incubated for 24 h, 48 h, and 72 h. Hypoxia was induced using 100 µM CoCl₂ or by placing the cells in a hypoxia induction chamber. Then, 13 μL of the CCK-8 reagent was added to each well for 3 h. Absorbance was measured at 450 nm using a multimode microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland). The results were quantified relative to the control, and they are presented as percentages. The half-maximal cell viability inhibition concentration (IC₅₀) and half-maximal growth inhibition concentration (GI₅₀) were determined using GraphPad Prism ver. 7.0 software (GraphPad Software, San Diego, CA, USA).