Telomere dysfunction is associated with cellular senescence in pig cells. (A) Morphology of adult fibroblasts (AF) during subculture by β-galactosidase staining. P, passage. Senescent cells are stained blue. (B) Quantification of senescent cells positive for β-galactosidase staining. (C) Relative expression levels by qPCR of the senescence-related genes, p53 and p21, in 3 types of pig cells during subculture. *, p < 0.05; **, p < 0.001, compared to the primary cells at the earliest passage. (D) P53 protein levels in different cell lines from early to late passages by immuno-blot analysis. β-actin served as loading control. (E) Representative images showing TIFs as DNA damage foci (white arrows) indicated by γ-H2AX foci at telomeres by IF-FISH. Nuclei, blue; Telomere, green; γ-H2AX, red. (F) Percentage of TIFs and γ-H2AX-positive nucleus in various pig cell types. In all, 100 cells were counted for each cell line. *, p < 0.05; **, p < 0.01, compared to the early passage of the same cell type.