Cell senescence assay

To assess senescence, β-galactosidase activity was measured with a histochemical staining kit (CST, USA) and performed according to the manufacturer’s instructions. Briefly, at 48h after transfection, remove the growth media from the cells. Rinse the plate one time with 1×PBS. Add 1mL of 1×Fixative Solution to each 35 mm well, allow cells to fix for 10–15 minutes at room temperature. Rinse the plate two times with 1×PBS. Add 1mL of the β-Galactosidase Staining Solution to each 35 mm well, seal plate with parafilm to prevent evaporation. Incubate the plate at 37°C overnight in a dry incubator (no CO2). While the β- Galactosidase Staining Solution is still on the plate, check the cells under a microscope for the development of blue color.