Fig. 5

Translated circ-FBXW7 suppressed stem cells’ renewal in a β-catenin ubiquitylation way. A Schematic image illustrated the IRES independent translation potential of circ-FBXW7. B Designation of junction probe and Flag-tag to detect translated circ-FBXW7-AA. C The specifically designed and synthesized FLAG antibody successfully detected circ-FBXW7-AA in 293 T cells. D The reader of YTHDF3 inhibition blocked m6A quantity, and decreased circ-FBXW7-AA level. Over-expressed circ-FBXW7 inhibited the proliferation of stem cells group (E), and stimulated the differentiated-orientation cells dividing (F) The tags of “negative” and “positive” are referring to marker of circ-FBXW7 solely, and the positive-circFBXW7 was set as the positive control. G The increased differentiated-orientation cells dividing, together with the increased asymmetric cells dividing, decreased the malignant behaviors. Similarly, YTHDF3 inhibition failed to stimulate the circ-FBXW7 dependent differentiated cells division, and helped to restore the symmetric cells dividing. H The selecting cells of FSC gate was grouped based on distribution patterns of phenotypic markers of circ-FBXW7/H3K27me3/ALDH1A1. I The cells with negative label (circ-FBXW7⁻/H3K27me3⁺/ALDH1A1⁺) showed much higher ability to form the spheres when assessing in per 100 cells, and the positive label (circ-FBXW7⁺/H3K27me3⁻/ALDH1A1⁻) greatly decreased the spheres forming ability. J Enforced circ-FBXW7 stimulated symmetric differentiation dividing, and consequently resulted in spheres forming inhibition. K The overexpressed circ-FBXW7 affected the downstream Wnt/β-catenin signaling (CCND1/CMYC) to conduct the renewal inhibition effects. L Co-immunoprecipitation assay revealed the endogenous circ-FBXW7-185AA could interact with endogenous β-catenin in both HCC827 cells and H1975 cells. Circ-FBXW7-185AA negatively regulated the protein stability of β-catenin (M), the ubiquitination status of β-catenin increased with overexpressed circ-FBXW7-185AA (N). O The ubiquitination level of β-catenin gradually increased with the overexpressed circ-FBXW7-185AA, and MG132 weakened the inhibitory effect of circ-FBXW7-185AA on β-catenin expression