Cell viability assay

Cells were treated with either vehicle (control), rapamycin (Sigma, 20 nmol/L, 5μM) [43], metformin (Enzo Life Sciences, 0, 12.5, 25, 50, 100, and 200μM), or rapamycin (Sigma) plus metformin for 12, 24, 36, and 48 h, respectively. One day prior to treatment, cells (1×10⁴) were seeded in 96-well plates. Appropriate amounts of the compounds were then added to achieve the indicated concentrations. Cell growth and proliferation were determined with a Cell Counting Kit-8 (Dojindo, Japan). After addition of 10 μl CCK-8 solution to each well, cells were incubated for 1.5 h, and absorbance was measured on a microplate reader (Spectramax 190, US) at 450 nm.