Figure 6.

Transforming growth factor-β1 (TGF-β1)–induced epigenetic repression of CXCL10 in fibroblasts from F-NL is mediated by an interdependent cross-talk between EZH2 and G9a. (A–F) F-NL cells were transfected with control siRNA, EZH2 siRNA, or G9a siRNA in culture medium for 72 hours before incubation with TGF-β1 (2 ng/ml) in culture medium for 48 hours and in serum-free medium for 24 h before (G) being treated with IL-1β (1 ng/ml) in the presence of TGF-β1 for a further 24 h. The protein–DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted. (A) EZH2, (B) H3K27me3, (C) G9a, (D) H3K9me3, acetylated histone (E) H3 and (F) H4, and total histone (B, D, and E) H3 and (F) H4 were immunoprecipitated with specific antibodies. The associated CXCL10 promoter DNA was amplified by real-time RT-PCR, and the amount was calculated and normalized to (A and C) input control or (B, D, E, and F) total histone H3 and H4. (G) CXCL10 concentration in the medium was analyzed by ELISA. Data are expressed as mean ± SEM of six separate experiments performed in duplicate. *P < 0.05, **P < 0.01 compared with control cells; ⁺P < 0.05, ⁺⁺P < 0.01 compared with TGF-β1 alone.