Fig. 7

circEIF3I promotes the recruitment of SMAD3 to early endosomes via binding with AP2A1. A RNA pull-down and western blot assays showed the interaction between circEIF3I and AP2A1 in PANC-1 and BxPC-3 cells. B Representative IF images identified the enrichment of AP2A1 on early endosomes (EEs) in PANC-1 cells. The red indicated AP2A1, the green indicated the EEA1 (marker of EEs), the blue (DAPI) indicated the nucleus (Scale bar = 10 μm). C The RNA immunoprecipitation (RIP) and qRT-PCR assays demonstrated specific binding of circEIF3I and AP2A1 (lower panel). IP efficiency of AP2A1 antibody shown by western blot (upper panel). D Representative FISH-IF images identified the colocalization of circEIF3I and AP2A1 in cytoplasm. The green (FAM-labelled probe) indicated the circEIF3I, the red indicated AP2A1, the blue (DAPI) indicated the nucleus (Scale bar = 10 μm). E and F Western blot analysis of indicated proteins in AP2A1 immunoprecipitated complexes or SMAD3 immunoprecipitated complexes from lysates of PANC-1 and BxPC-3 with circEIF3I KD, which were treated with 5 ng/ml TGF-β for 15 min. G Western blot showed the p-SMAD3 expression in PANC-1 and BxPC-3 cells transfected with sh-ctrl or sh-circEIF3I-2, with or without AP2A1 KD (5 ng/ml TGF-β, 15 min). H The binding of SMAD3 and TGFβR I in PANC-1 transfected with sh-ctrl or sh-circEIF3I-2, with the ectopic expression of circEIF3I-WT, circ-mut-SMAD3 and circ-mut-AP2A1 respectively in circEIF3I KD groups (5 ng/ml TGF-β, 15 min). I The p-SMAD3 expression in PANC-1 and BxPC-3 cells transfected with sh-ctrl or sh-circEIF3I-2, with the ectopic expression of circEIF3I-WT, circ-mut-SMAD3 and circ-mut-AP2A1 respectively in circEIF3I KD groups (5 ng/ml TGF-β, 15 min). J RNA-EMSA determined the specific binding between SMAD3 and biotin-labelled Probe3 of circEIF3I, with SMAD3 antibody incubation (super shift band). Data are shown as the mean ± SD of three replicates; **P < 0.01