Fig. 6.

lncRNA-CD244 interacts directly with EZH2 and recruits EZH2 to ifng and tnfa promoters. (A) Gel electrophoresis of lncRNA-CD244 extracted from nucleus and cytoplasm of CD8⁺ T cells purified from PBMCs of patients with active TB. As controls, more actin B expressed in cytoplasm and more U6 expressed in nucleus, respectively. (B) IB analysis of EZH2 in the IP by IgG or anti-EZH2–specific antibody from CD8⁺ T-cell lysates of patients with active TB. (C and D) Gel electrophoresis (C) and qPCR analysis (D) of lncRNA-CD244 retrieve in IP by IgG or anti-EZH2–specific antibody from CD8⁺ T-cell lysates of patients with active TB. The levels of qRT-PCR products were expressed as a percentage of input RNA. (E) Biotinylated lncRNA-CD244 or antisense RNA control was incubated with nuclear extracts of CD8⁺ T cells from patients with active TB and targeted with streptavidin-conjugated magnetic beads (MB), and associated proteins were assessed with Western blot using anti-EZH2–specific antibody. (F) Confocal microscopic images of RNA FISH assay of lncRNA-CD244 and immunofluorescence analysis of EZH2 show that EZH2 colocalizes with lncRNA-CD244 in nucleus of CD8⁺ T cells from patients with active TB. Lower images were cropped from the squares in the upper images. (Scale bars: 10 μm in Upper and 5 μm in Lower.) More than 30 cells were examined and had similar results. White arrowheads mark the EZH2/lncRNA-CD244 colocalization. (G and H) ChIP-qPCR analysis of WDR5, G9a, Prdm16, EZH2, and control antibodies at the promoters of IFN-γ and TNF-α in CD8⁺ T cells transfected with siRNA-lncRNA and siRNA-Ctrl (n = 7). ***P < 0.001. Error bars represent SEM from three independent experiments.