Fig. 4

CircGSK3B interplayed with EZH2 protein in GC cells. a Coomassie bright blue staining (left panel), mass spectrometry analysis, as well as overlapping assays (Venn diagram, right panel) with developed RBP and TF databases suggesting proteins pulled down based on biotin-labeled antisense or sense forms of circGSK3B from the lysates of MKN45 cells. b RIP and real-time RT-qPCR assays were used to assess the relative interplay between circGSK3B and 9 proteins in MKN45 cells with circGSK3B or linear GSK3B, which were normalized to GC cells transfected using circ-Mock. c RIP assay with primer datasets revealing the interaction between circGSK3B and EZH2/EED/SUZ12 in MKN45 cells with circ-Mock, circGSK3B, or lin-GSK3B. d MS assay showing the EZH2 peptides pulled down by circGSK3B. e Dual RNA-FISH and immunofluorescence staining assays indicating the co-localization of circGSK3B (green) and EZH2 (red) in AGS and MKN-45 cells, with nuclei staining using DAPI (blue). f Schematic diagram indicating the domains of EZH2 truncations. g In vitro binding assay showing the enriched circGSK3B levels assessed based on RT-PCR (lower panel) after incubation using full-length or truncations versions of Flag-tagged or GST-tagged recombinant EZH2 protein validated by western blot (upper panel)