Fig. 3
circPDE5A binds to WTAP and regulates its m6A methylation activity. A, Identification of circPDE5A binding proteins using silver staining. B, Detection of the interaction between circPDE5A and m6A methyltransferases or demethylases through western blotting. C, The binding capacity between circPDE5A and WTAP in C4-2B and 22Rv-1 cells through RNA-IP assay. D, IF and FISH assays showing the colocalization of WTAP and circPDE5A in C4-2B and 22Rv-1 cells. Scale bars, 5 μm. E, Analysis of the expression of circPDE5A in 22Rv-1 cells with WTAP overexpression or knockdown through RT-qPCR. F, Analysis of the expression of WTAP in 22Rv-1 cells with circPDE5A overexpression or knockdown by western blot. G, Analysis of the cellular distribution of WTAP in 22Rv-1 cells with circPDE5A overexpression or knockdown by western blot. H, Analysis of the cellular distribution of WTAP in 22Rv-1 cells with circPDE5A overexpression or knockdown through immunofluorescence. Scale bars, 10 μm. I, Dot blot showing the total m6A modification level in 22Rv-1 cells with circPDE5A overexpression or knockdown. J, Immunofluorescence showing the total m6A modification level in 22Rv-1 cells with circPDE5A overexpression or knockdown. Scale bars, 10 μm. K, Co-IP assay showing the binding capacity between WTAP and METTL3 or METTL14 in 22Rv-1 cells after circPDE5A overexpression or knockdown. L, M, Transwell assay showing the migration and invasion ability with cells subjected to WTAP knockdown (L) or overexpression (M) in 22Rv-1 cells. Scale bars, 5 μm. Data represents mean ± S.D. from three independent experiments. ***, p < 0.001