Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

doi: 10.1165/rcmb.2017-0286OC

Figure Lengend Snippet: EZH2 and G9a inhibition and knockdown promote histone acetylation at the CXCL10 promoter in F-IPF. F-IPF cells were (A and B) incubated with DZNep (10 nM) or (C and D) transfected with control siRNA, G9a siRNA, or EZH2 siRNA in culture medium for 48 hours and in serum-free medium for 24 hours before being treated with IL-1β (1 ng/ml) for a further 4 hours. The protein–DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted. Acetylated and total histone (A and C) H3 and (B and D) 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 total histone H3 and H4. Data are expressed as mean ± SEM of experiments with six separate F-IPF cell lines performed in duplicate. *P < 0.05 compared with corresponding untreated cells.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express Kit and the Re-ChIP-IT Express Kit (Active Motif), as described elsewhere ( 5 , 10 ), with antibodies against H3K27me3, H3K9me3, EZH2, acetylated and total H3 and H4 (Merck Millipore), and G9a (Santa Cruz Biotechnology).

Techniques: Inhibition, Knockdown, Incubation, Transfection, Control, Immunoprecipitation, Amplification, Quantitative RT-PCR

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

doi: 10.1165/rcmb.2017-0286OC

Figure Lengend Snippet: 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.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express Kit and the Re-ChIP-IT Express Kit (Active Motif), as described elsewhere ( 5 , 10 ), with antibodies against H3K27me3, H3K9me3, EZH2, acetylated and total H3 and H4 (Merck Millipore), and G9a (Santa Cruz Biotechnology).

Techniques: Transfection, Control, Incubation, Immunoprecipitation, Amplification, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

doi: 10.1165/rcmb.2017-0286OC

Figure Lengend Snippet: EZH2 and G9a knockdown alters repressive histone methylation at the CXCL10 promoter in F-IPF. F-IPF cells were transfected with control siRNA, EZH2 siRNA, or G9a siRNA before being treated with IL-1β (1 ng/ml) for a further (A) 24 hours or (B and C) 4 hours. (A) Total cell lysates were collected for Western blot analysis of EZH2 and G9a with β2M as the loading control. This is representative of three separate experiments with different F-IPF cell lines. Chromatin immunoprecipitation was conducted with specific antibodies against (B) H3K27me3, (C) H3K9me3, and (B and C) total histone H3. (B and C) The associated CXCL10 promoter DNA was amplified by real-time RT-PCR, and the amount was calculated and normalized to total histone H3. Data are expressed as mean ± SEM of experiments with six separate F-IPF cell lines performed in duplicate. *P < 0.05, **P < 0.01 compared with corresponding untreated cells.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express Kit and the Re-ChIP-IT Express Kit (Active Motif), as described elsewhere ( 5 , 10 ), with antibodies against H3K27me3, H3K9me3, EZH2, acetylated and total H3 and H4 (Merck Millipore), and G9a (Santa Cruz Biotechnology).

Techniques: Knockdown, Methylation, Transfection, Control, Western Blot, Chromatin Immunoprecipitation, Amplification, Quantitative RT-PCR

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

doi: 10.1165/rcmb.2017-0286OC

Figure Lengend Snippet: EZH2 and G9a interact with each other to regulate histone methylation at the CXCL10 promoter in F-IPF. F-IPF cells were (A–D) transfected with control siRNA, G9a siRNA, or EZH2 siRNA or (E and F) untreated. Chromatin pellets were (A–F) extracted and immunoprecipitated with specific antibodies against (A and F) EZH2, (C and E) G9a, (B) H3K27me3, (D) H3K9me3, and (B and D) total histone H3. The immunoprecipitates (IPs) of (E) G9a and (F) EZH2 were immunoprecipitated again with antibodies against (E) EZH2 and (F) G9a. The associated CXCL10 promoter DNA was amplified by real-time RT-PCR, and the amount was calculated and normalized to (B and D) total histone H3 or (A, C, E, and F) input control. Data are expressed as mean ± SEM of experiments with six separate F-IPF cell lines performed in duplicate. **P < 0.01 compared with untreated cells. Serum-starved F-IPF cells grown on sterile glass coverslips were fixed in 4% paraformaldehyde. In situ interaction between EZH2 and G9a was detected by proximity ligation assay with primary mouse anti-EZH2 antibody and rabbit anti-G9a antibody as well as by proximity ligation assay probes Anti-Mouse MINUS and Anti-Rabbit PLUS. (G) Amplified DNA was detected by Duolink In Situ Detection Reagents Red, and DAPI (blue) was used to identify the nuclei. Images are representative of three separate experiments with different F-IPF cell lines. Scale bars: 20 μm.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express Kit and the Re-ChIP-IT Express Kit (Active Motif), as described elsewhere ( 5 , 10 ), with antibodies against H3K27me3, H3K9me3, EZH2, acetylated and total H3 and H4 (Merck Millipore), and G9a (Santa Cruz Biotechnology).

Techniques: Methylation, Transfection, Control, Immunoprecipitation, Amplification, Quantitative RT-PCR, Sterility, In Situ, Proximity Ligation Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

doi: 10.1165/rcmb.2017-0286OC

Figure Lengend Snippet: EZH2 and G9a inhibition and knockdown promote histone acetylation at the CXCL10 promoter in F-IPF. F-IPF cells were (A and B) incubated with DZNep (10 nM) or (C and D) transfected with control siRNA, G9a siRNA, or EZH2 siRNA in culture medium for 48 hours and in serum-free medium for 24 hours before being treated with IL-1β (1 ng/ml) for a further 4 hours. The protein–DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted. Acetylated and total histone (A and C) H3 and (B and D) 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 total histone H3 and H4. Data are expressed as mean ± SEM of experiments with six separate F-IPF cell lines performed in duplicate. *P < 0.05 compared with corresponding untreated cells.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express Kit and the Re-ChIP-IT Express Kit (Active Motif), as described elsewhere ( 5 , 10 ), with antibodies against H3K27me3, H3K9me3, EZH2, acetylated and total H3 and H4 (Merck Millipore), and G9a (Santa Cruz Biotechnology).

Techniques: Inhibition, Knockdown, Incubation, Transfection, Control, Immunoprecipitation, Amplification, Quantitative RT-PCR

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

doi: 10.1165/rcmb.2017-0286OC

Figure Lengend Snippet: EZH2 and G9a inhibition and knockdown restore CXCL10 expression in F-IPF. F-IPF cells were (A and B) incubated with DZNep (10 nM) or (C and D) transfected with empty vector, pMSCV-mJMJD3, or pMSCV-JMJD3 or (E and F) transfected with control siRNA, EZH2 siRNA, or G9a siRNA in culture medium for 48 hours and in serum-free medium for 24 hours before being treated with IL-1β (1 ng/ml) for a further (A, C, and E) 4 hours or (B, D, and F) 24 hours. Total RNA was isolated, and mRNA levels of CXCL10 and the internal control β2M were determined by real-time RT-PCR. (A, C, and E) Results are calculated as the ratio of CXCL10 mRNA and β2M mRNA. (B, D, and F) Medium was collected, and CXCL10 concentration 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 corresponding untreated cells.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express Kit and the Re-ChIP-IT Express Kit (Active Motif), as described elsewhere ( 5 , 10 ), with antibodies against H3K27me3, H3K9me3, EZH2, acetylated and total H3 and H4 (Merck Millipore), and G9a (Santa Cruz Biotechnology).

Techniques: Inhibition, Knockdown, Expressing, Incubation, Transfection, Plasmid Preparation, Control, Isolation, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

doi: 10.1165/rcmb.2017-0286OC

Figure Lengend Snippet: 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.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express Kit and the Re-ChIP-IT Express Kit (Active Motif), as described elsewhere ( 5 , 10 ), with antibodies against H3K27me3, H3K9me3, EZH2, acetylated and total H3 and H4 (Merck Millipore), and G9a (Santa Cruz Biotechnology).

Techniques: Transfection, Control, Incubation, Immunoprecipitation, Amplification, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis

doi: 10.1165/rcmb.2017-0286OC

Figure Lengend Snippet: A hypothetical model depicting the role of the interplay between EZH2 and G9a in the regulation of CXCL10 gene repression in idiopathic pulmonary fibrosis. EZH2 and G9a physically and interdependently interact with each other in F-IPF. EZH2- and G9a-mediated H3K27me3 and H3K9me3 result in the recruitment of HDACs to the CXCL10 promoter. This then leads to histone deacetylation, causing reinforced epigenetic silencing of the CXCL10 gene in F-IPF. Inhibition or depletion of either EZH2 or G9a leads to the removal of repressive H3K27me3, H3K9me3, and histone deacetylation, resulting in chromatin derepression and CXCL10 gene reexpression in F-IPF.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express Kit and the Re-ChIP-IT Express Kit (Active Motif), as described elsewhere ( 5 , 10 ), with antibodies against H3K27me3, H3K9me3, EZH2, acetylated and total H3 and H4 (Merck Millipore), and G9a (Santa Cruz Biotechnology).

Techniques: Inhibition