Fig. 2

NOS1 inhibits interferon-induced recruitment of HDAC2. a Negative control siRNA (si-NC) or an siRNA specific for HDAC2 (si-HDAC2) was used to transfect A375 cells for 48 h, and then the knockdown efficiency was determined by RT-PCR and an immunoblotting assay. b, c siRNA was used to transfect b A375 cells and c SW480 cells for 24 h, followed by stimulation with IFNα (1000 U/ml) for 6 h and RT-PCR analysis. d HDAC2 expression plasmids were used to transfect A375 cells for 24 h, and the transfection efficiency was determined by immunoblotting assay. e Similar to b, HDAC2 expression vectors were used to transfect A375 cells for 24 h before IFNα stimulation. f pISRE-luc and Renilla-luc reporter plasmids were used to cotransfect A375 cells in the presence of si-NC (Con), si-HDAC2 (HD2), or HDAC2 expression vectors as indicated. Luciferase activity was measured 24 h after transfection in untreated cells and cells treated with IFNα (1000 U/ml) for 6 h. g A375 cells were incubated with or without IFNα (1000 U/ml, 6 h). ChIP-qPCR was used to detect the binding of HDAC2 to the ISG promoters. h A375 cells were stimulated with (+) or without (−) IFNα (1000 U/ml) for 6 h, and the lysates were precipitated with HDAC2 or IgG antibodies. Western blotting was performed using the indicated antibody. i Similar to h, but Co-IP assays were performed in Control/NOS1 (A375) cells. j Similar to g, ChIP assays were performed in Control/NOS1 (A375) cells by stimulation with IFNα (1000 U/ml) for 6 h. k, l Control/NOS1 (A375) cells were treated with IFNα (1000 U/ml) for 6 h and 12 h, the expression of HDAC2 was detected by k western blotting and l RT-PCR. m The subcellular localization of HDAC2 was visualized by immunofluorescence staining in A375 cells after treatment with or without GSNO for 6 h. Nuclei were stained with DAPI (blue), original magnification: 100×. n A375 cells were treated with or without IFNα (1000 U/ml) for 12 h, and the expression of nuclear and cytoplasmic proteins of HDAC2 was detected by western blotting. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Flag-HD2, Flag-tagged HDAC2 expression vectors; End-HD2, endogenous HDAC2