Fig. 2
IGF2BPs preferentially bind DNA: RNA hybrids containing m6A-modified RNA and overexpression of IGF2BPs causes R-loop accumulation. A Normalized abundance of eluted pull-down proteins using RNA: DNA hybrid probes with methylated (red) or unmethylated (green) adenosine. B Identification of m6A-RNA: DNA hybrid specific binding proteins by RNA pull-down. Silver staining (upper left) and western blot (upper right) indicated pulldown of proteins from PC-3 nuclear extract. C Western blot results showed that IGF2BP proteins preferentially bind the RNA: DNA hybrids containing m6A-modified RNA. D Co-localization of m6A with R-loops in PC-3 cells. Scale bar = 60 μm. E Quantification of cellular immunofluorescence (n = 17 per group). F DU-145 and PC-3 cells were transfected with Myc-IGF2BP1/2/3 and vector. The R-loop levels were evaluated by dot-blot, and RNaseH1 treated samples were used as negative control. G-H PC-3 cells were treated with m6A demethylation drug (DAA, 3-Deazaadenosine) and transfected with Myc-IGF2BP1/2/3 and vector. The R-loop levels (Left of Fig. G) and m6A levels (H) are shown. (Right of Fig. G) Quantification of dot blot results in Fig. G. Data are presented as means ± SD, two-tailed unpaired t-test. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001