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. 2015 Dec 1;16(12):1215–1227. doi: 10.1038/ni.3279

Figure 4. PARP9-DTX3L interacts with STAT1 and enhances translocation of STAT1 to the nucleus and its binding to ISGs and activation of ISG promoters.

Figure 4

(a) Immunoblot analysis of the co-immunoprecipitation of FLAG-tagged STAT1 (left and middle) or STAT1-CC (right) with PARP9 or DTX3L (left margin) in parental U3A cells (left) or U3A cells expressing FLAG-tagged STAT1 (middle) or STAT1-CC (right), with (γ or β) or without (−) treatment for 24 h with IFN-γ (100 U/ml) or IFN-β (1,000 U/ml) (above lanes). (b) Immunoblot analysis of tyrosine-phosphorylated STAT1 (pY-STAT1), serine-phosphorylated STAT1 (pS-STAT1), or total STAT1, PARP9 or DTX3L (left margin) in cytosolic (C) and nuclear (N) fractions of U3A, U3A-STAT1 or U3A-STAT1-CC cells with or without treatment for 0.3 h with IFN-γ (as in a). Dashed outlines indicate tyrosine-phosphorylated STAT1, PARP9 and DTX3L in the nuclear fraction. (c) Immunostaining of U3A-STAT1-PARP9-DTX3L cells with (+ LMB) or without treatment for 1 h with leptomycin B (10 ng/ml), then with (+ IFN-β) or without (− IFN-β) treatment for 0.3 h with IFN-β (1,000 U/ml) plus leptomycin B. Scale bar, 10 μm. (d) Crosslinking ChIP assay of the binding of STAT1 to the IRF1 promoter in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells left untreated (− IFN-β) or treated (+ IFN-β) for 0.3 h with IFN-β; after incubation of lysates with antibody to STAT1, the immunoprecipitated DNA was analyzed by real-time PCR with primers for IRF1 promoter, then results were normalized to those of input DNA. Results obtained with control antibody (mouse immunoglobulin G (IgG)) were similar to those of cells transfected with empty vector only (data not shown). (e) Transactivation of ISRE and GAS in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells transfected for 12 h with a luciferase reporter vector for ISRE (left) or GAS (right) and left untreated (−) or treated (+) with IFN-β (left) or IFN-γ (right); results are presented as luminescence units (LU) relative to the activity of renilla luciferase. (f) Translocation of STAT1 to the nucleus in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells before (−) and after (+) treatment for 0.3 h with IFN-β (1,000 U/ml) (left) or IFN-γ (100 U/ml) (right), presented as fluorescence units (FU) (nuclear fluorescence − cytoplasmic fluorescence); initial negative values (for −IFN-β) indicate that STAT1 was mainly cytoplasmic. *P < 0.01, versus U3A-STAT1 cells (unpaired t-test). Data are representative of three independent experiments (mean and s.e.m. (d,e) or mean and s.e.m. of three wells with 500 cells per well (f)).