Fig. 5

Direct interaction between circPTEN1 and Smad4 inhibits the TGF-β1/Smad signaling pathway. A. Left, schematic illustrating the cyclization of linear RNA generated in vitro. Right, top, RT-PCR analysis of linear and cyclized circPTEN1 RNAs. Right, bottom, RNA-protein pull-down assays were conducted with FLAG-Smad4 purified from 293 T cells transfected with pCMV-tag-2b-Smad4 against cyclized circPTEN1. B. Electrophoretic mobility shift analysis of interactions between circPTEN1 and FLAG-Smad4. C. The Smad4 protein is characterized by the presence of Mad homology domain-1 (MH1) and Mad homology domain-2 (MH2). D. RIP assays showing the association of full-length Smad4 or Smad4 truncations with circPTEN1. Top, IP efficiency of FLAG-antibody shown by western blot. Down, relative enrichment representing RNA levels associated with full-length Smad4 or Smad4 truncations relative to an input control. IgG antibody served as a control. E. Electrophoretic mobility shift analysis of interactions between circPTEN1 and FLAG-Smad4 or FLAG-Smad4 truncations. F. The distribution of Smad2, Smad3, p-Smad2 and p-Smad3 in the nuclear fraction. LoVo cells with circPTEN1 knockdown or overexpression as indicated in Supplementary Fig. 6C and 6E were treated with 5 ng/mL TGF-β1 for 1 h. Nuclear proteins were extracted to detect the expression of the indicated proteins. Lamin B was used as a nuclear marker. RS: pLCDH-circPTEN1-RS. G. Subcellular localization of Smad2 or Smad3. C-terminal GFP-tagged Smad2 or Smad3 was introduced into circPTEN1 knockdown or overexpression LoVo cells, prior to be treated with 5 ng/mL TGF-β1 for 1 h. Cells were then stained with DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm. RS: pLCDH-circPTEN1-RS. H. circPTEN1 inhibits the formation of Smad2-Smad4 and Smad3-Smad4 complexes. Western blot analysis of Smad2 and Smad3 following immunoprecipitation of Smad4 from circPTEN1 knockdown or overexpression LoVo cells as indicated in Supplementary Fig. 6C and 6E, which were treated with 5 ng/mL TGF-β1 for 1 h. RS: pLCDH-circPTEN1-RS