Fig. 3: KDM2A induces p53-dependent senescence in normal human mammary fibroblasts.

a Senescence of normal mammary fibroblasts RMF-EG cells co-cultured without or with breast cancer cells (MCF-7 or MDA-MB-231) and KDM2A-expressing K2A-1 and K2A-2 cells was investigated by β-galactosidase staining. The percentage of senescent cells of control RMF-EG, K2A-1 and K2A-2 was also quantified (n = 3). b The expression of senescence-associated secretary phenotype cytokines including TGF-β, IL-6, IL-8 and CXCL1 was detected by quantitative RT-PCR (n = 3). c The p53 protein level and the phosphorylation of Chk2 and p38 of KDM2A-overexpressing K2A-1 cells were compared to that of normal RMF-EG mammary fibroblasts. The phosphorylation of H2AX protein (γ-H2AX) was investigated by immunofluorescent staining and the number of γ-H2AX foci of RMF-EG and K2A-1 cells was compared (n = 3). d RMF-EG cells were pretreated without or with daminozide (2.5 μM) and stimulated with IL-6 (20 ng/ml) for 24 h. The mRNA and protein levels of p53 were investigated by quantitative RT-PCR and western blotting (n = 3). e RMF-EG cells were treated as described above and the expression of CXCL1 and IL-8 was quantified by quantitative RT-PCR (n = 3). f The K2A-1 cells were transfected with p53 siRNA (p53i) or control siRNA (Ci). After 24 h, cellular mRNAs and proteins were harvested. The protein level of KDM2A, p53 and β-galactosidase (GLB1) was investigated by western blotting and the mRNA level of IL-8 and CXCl1 was studied by quantitative RT-PCR (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.