Senescence associated β-galactosidase (SA-β gal) assay

The senescence-associated β-galactosidase activity was detected using the SA-β-gal staining kit (Cell signaling, Danvers, USA) according to the manufacturer’s instructions. Briefly, cells were washed with PBS and fixed for 10–15 min in 1X fixative solution at room temperature. After washing with PBS, the cells were incubated overnight at 37 °C with 1X SA-β-gal staining solution (pH 6.0). The percentage of positive cells and cell size were analyzed with a microscope via a high and digital camera (Nikon instrument, Nikon Inc., Melville, NY, USA) and image J program (NIH). Positive signals of SA-b-gal staining were marked by an arrowhead.