Galactose-Based Screening for Cell Viability

Drug screening was assessed by culturing the cells in a restrictive medium with galactose as the unique carbon source. The galactose medium was prepared using DMEM no glucose (Invitrogen^TM Molecular Probes, Eugene, OR, USA) supplemented with 20 mM D-galactose, 15 mM HEPES, 1% penicillin/streptomycin, and 10% FBS. Cells were seeded in 24-well plates in DMEM 1 g/L glucose and treated with different compounds. After 3 days, the glucose medium was removed and changed to galactose medium, with treatments reapplied. Images and cell counting were obtained immediately (T0) and 72 h after the shift to galactose medium (T72) using the BioTek^TM Cytation 1 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). The proliferation ratio was obtained by dividing the number of cells at T72 by the number of cells at T0. Proliferation ratio values above 1 were considered as cell proliferation, while values below 1 were considered as cell death, and a value of 1 indicated cell survival. Positive treatments were those that allowed the survival of patient cells in the glucose-free galactose medium, with the cocktail of polydatin and nicotinamide at 10 µM selected, as the others failed to make mutant cells survive and were deemed negative. Cell viability was confirmed by trypan blue dye exclusion.
This screening was repeated using 3-TYP, a specific inhibitor of SIRT3. The concentration employed was 32 nM, as this compound exhibits an IC₅₀ (half-maximal inhibitory concentration) of 16 nM for SIRT3, requiring a higher concentration for SIRT1 (IC₅₀ = 88 nM) and SIRT2 (IC₅₀ = 92 nM). This ensures the specific inhibition of SIRT3 without affecting other sirtuins. Cells were initially seeded in glucose medium and treated with polydatin and nicotinamide at 10 µM, along with 3-TYP at 32 nM for a duration of 3 days. Subsequently, the glucose medium was replaced with galactose medium, treatments were renewed, and images were captured using the same methodology as in the previous screening.