Figure 11. Effects of Wnt/β-catenin signaling on γ-H2A.X and p16^INK4a expression.

(A) Immunofluorescence staining of γ-H2A.X. The expression of γ-H2A.X was hardly detected in the YRS group, whereas the number of γ-H2A.X–positive cells and γ-H2A.X expression level were markedly increased in the ORS group. In the ORS + DKK1 and ORS + si-β-catenin groups, the number of γ-H2A.X–positive cells and γ-H2A.X expression levels were clearly decreased. The pictures on the right are magnified images of the white box. γ-H2A.X expression is indicated by a stippled appearance in the nucleus. Green, γ-H2A.X; blue, DAPI. Scale bar  = 25 µm. (B) Western blot analysis of γ-H2A.X expression. β-Actin was used as the internal control. (C) Quantification of γ-H2A.X protein expression levels. γ-H2A.X expression was significantly increased in the ORS groups compared with that in the YRS groups (**P<0.01). After treatment with DKK1 or si-β-catenin in ORS, γ-H2A.X expression was markedly decreased compared with the ORS group (^## P<0.01; n = 5). (D) RT-PCR analysis of p16^INK4a expression. β-Actin was used as the internal control. (E) Quantification of p16^INK4a mRNA expression level. p16^INK4a expression in the ORS group markedly increased compared with that in the YRS group (**P<0.01). However, in the ORS + DKK1 and the ORS + si-β-catenin groups, the p16^INK4a mRNA levels were significantly decreased compared with the ORS groups (^# P<0.05 or ^{# #} P<0.01; n = 5).