Fig. 7

EIF4A3 binds Smad2/3 mRNA directly. (A) The putative binding sites between EIF4A3 protein and Smad2/3 mRNA were predicted by bioinformatics analysis (Starbase). (B) The direct interaction between EIF4A3 protein and Smad2/3 mRNA was assessed by RIP assay. Normal IgG served as a negative control. (C) The co-localization of Smad2/3 mRNA and EIF4A3 protein was assessed by RNA FISH and IF staining. Green, Smad2/3 mRNA; Red, EIF4A3; Blue, DAPI. Scale bar, 20 μm. (D) Correlation of Smad2/3 expression with overall survival of CRC patients was from GEPIA database. (E) The mRNA levels of Smad2 and Smad3 in CRC tissues were determined by qRT-PCR. (F) Pearson correlation analysis between EIF4A3 and Smad2, as well as between EIF4A3 and Smad3 in CRC tissues. (G) The mRNA levels of EIF4A3 and Smad2/3 in HUVECs were detected by qRT-PCR. (H) The stabilities of Smad2/3 mRNA were assessed by RNA stability assay. (I) The protein levels of EIF4A3 and p-Smad2, Smad2, p-Smad3, and Smad3 were detected by western blot. *, P < 0.05, **, P < 0.01, ***, P < 0.001