Figure 6: RNA–DNA triplexes are present in vivo.

(a) Confocal microscopic images showing immunostaining with anti-triplex dA.2rU antibody (green) in BT-549 cells. The nucleus is stained with DAPI (4,6-diamidino-2-phenylindole; blue). Immunostaining with no antibody and secondary antibody were used as negative controls. Scale bar, 5 μm. The graph to the right shows quantification of the triplex signal in cytoplasm and nuclear compartments obtained from the three-dimensional confocal images. The graph represents the average of cytoplasmic and nuclear signals from >50 cells in several microscopic fields. The error bars indicate s.e.m. The P value was calculated using Student’s t-test **P<0.01. (b) RNA–DNA triplex structures are sensitive to RNase A but are resistant to RNase H in vivo. Top panel: immunofluorescent staining of BT-549 cells with anti-triplex dA.2rU antibody (green) with no treatment (left), pretreated with RNase A (centre), or pretreated with RNase H (right) as indicated. Middle panel: cells were counterstained with DAPI (blue). Bottom panel: overlay of the triplex signals with DAPI staining. Scale bar, 5 μm. (c) Triplex-ChIP–qPCR showing enrichment (presented as percentage of input) of triplex structures over the MEG3 peaks associated with the TGF-β pathway genes (TGFBR1, TGFB2 and SMAD2) in BT-549 cells (±s.d., n=3). Actin was used as a negative control. Chromatin was pretreated with RNase A or RNase H before ChIP. Immunoglobulin G (IgG) was used as an antibody control. (d) Triplex-ChIP–qPCR showing enrichment (presented as percentage of input) of triplex structures over the MEG3 peaks associated with the TGF-β pathway genes (TGFBR1, TGFB2 and SMAD2) in Ctrlsh and MEG3sh BT-549 cells (±s.d., n=3). IgG was used as an antibody control.