Annexin 5 pi staining

All assays were performed as previously described (31 (link)). Briefly, cells were plated in triplicate wells of 6-well dishes at a low density (150 cells/well) and cultured under normal conditions without perturbation. After 10 days, the colonies were washed with PBS and stained with crystal violet (0.5% w/v in 25% methanol). Stained plates were rinsed in ddH₂O and allowed to dry at room temperature. The plates were photographed, and colonies were counted using ImageJ 1.8.0 software (NIH; National Institutes of Health, Bethesda, MD, USA). For the proliferation assay, cells (3×10³ per well) were cultured in 6-well plates in medium containing complete supplements. Cell proliferation was detected on a hemocytometer by cell counting every 3 days. For Annexin V/PI staining, cells (1×10⁵ per well) were cultured in 6-well plates with or without serum for 24 h and subjected to Annexin V/PI staining on the basis of the manufacturer's instructions (Vazyme, Nanjing, China) and immediately analyzed by flow cytometry.