Fig. 5

circEIF3I directly binds to SMAD3 and increases SMAD3 phosphorylation. A Schematic of MS2-GST protein-mediated capture of circEIF3I-MS2 from cellular lysates of overexpressing PANC-1 cell lines. B MS2-TRAP assay precipitated proteins were detected by silver staining. C The RNA immunoprecipitation (RIP) and qRT-PCR assays demonstrated specific binding of circEIF3I and SMAD3 (lower panel). IP efficiency of SMAD3 antibody shown by western blot (upper panel). D RNA pull-down assay showed the interaction between circEIF3I and SMAD3 in PANC-1 and BxPC-3 cells. E Representative FISH-IF images identified the colocalization of circEIF3I and SMAD3 in cytoplasm. The green (FAM-labelled probe) indicated the circEIF3I, the red indicated SMAD3, the blue (DAPI) indicated the nucleus (Scale bar = 10 μm). F RIP demonstrated the specific interaction between circEIF3I and MH2 domain of SMAD3. G Western blot analysis showed that circEIF3I retrieved HA-tagged full-length and SMAD3 MH2 domain using RNA pull-down assays. H Western blot showed the expression of p-SMAD3 and SMAD3 in cells with circEIF3I KD and overexpression, with the TGF-β treatment (5 ng/ml, 15 min). I Representative ISH and IHC images identified the expression of circEIF3I, p-SMAD3, MMP2, MMP9 and MMP14 derived from human PDAC tissue microarray (Scale bar = 100 μm). J Correlation analysis of histochemistry score (H-score) of circEIF3I with p-SMAD3, MMP2, MMP9 and MMP14, respectively. Data are shown as the mean ± SD of three replicates; ***P < 0.001