Figure 1.

Histone H3 lysine 27 trimethylation (H3K27me3) is increased and modulated at the CXCL10 promoter in fibroblasts from idiopathic pulmonary fibrosis lungs (F-IPF). (A and B) Confluent and serum-starved fibroblasts from nonfibrotic lungs (F-NL) and F-IPF cells were treated with IL-1β (1 ng/ml) for the times indicated. F-IPF cells were incubated with (C) 3-deazaneplanocin A (DZNep) (10 nM) or (D and E) transfected with empty vector, pMSCV-mJMJD3, or pMSCV-JMJD3 before being treated with IL-1β (1 ng/ml) for a further (C and E) 4 hours or (D) 24 hours. Chromatin immunoprecipitation was conducted with specific antibodies against (A, C, and E) H3K27me3, (B) enhancer of zest homolog 2 (EZH2), and (A, B, C, and E) total histone H3. The associated CXCL10 promoter DNA was amplified by real-time RT-PCR, and the amount was calculated and normalized to (A, C, and E) total histone H3 or (B) input control. Data are expressed as mean ± SEM of experiments with six separate F-NL and/or F-IPF cell lines performed in duplicate. *P < 0.05, **P < 0.01 compared with corresponding F-NL or untreated F-IPF cells. (D) Total cell lysates were collected for Western blot analysis of mutant variant of jumonji domain-containing protein 3 (mJMJD3) and jumonji domain-containing protein 3 (JMJD3) overexpression with β₂-microglobulin (β2M) as the loading control. This is representative of three separate experiments with different F-IPF cell lines.