Fig 2: CircRAPGEF5 binds to RBFOX2 protein.(A) RNA pull-down was performed using the biotin-labeled sense or antisense probes in the KLE cell. Bound proteins were analyzed by SDS–PAGE and silver staining. (B) RBFOX2 immunoblot analysis of sense and antisense circRAPGEF5 RNA pull-downs in KLE and Ishikawa cells. GAPDH was used as the loading control. (C) RIP assays in KLE and Ishikawa cells using RBFOX2 and IgG antibody. The precipitate was subjected to western blotting with antibodies against the indicated proteins. The relative levels of circRAPGEF5 enriched by RBFOX2 or IgG were determined by qRT-PCR. (D) FISH combined with IF staining were performed to determine colocalization of circRAPGEF5 and RBFOX2. (E) The catPAPID algorithm was used to predict the circRAPGEF5-RBFOX2 interaction. Schematic diagram illustrating full-length RBFOX2 and truncated forms of RBFOX2. (F) KLE cells were transfected with 3 × Flag-RBFOX2 full-length or truncations, which were determined by Western blot with an anti-FLAG antibody. (G) The relative enrichment of endogenous circRAPGEF5 in truncated RBFOX2 RIP was measured by qRT-PCR. Data were mean ± SD. ns, not significant, ***P < 0.001 (Student's t-test).

Fig 3: CircRAPGEF5 confers EC cells insensitivity to ferroptosis through regulating the alternative splicing of TFRC(A) Schematic diagram of TFRC splicing variants. The red box represents the alternative exon-4. (B) Cell viability was analyzed using a CCK-8 kit in Ishikawa cells transfected with empty vector control, circRAPGEF5, TFRC-L, TFRC-S, circRAPGEF5 + TFRC-L, ircRAPGEF5 + TFRC-S.(C) Lipid peroxidation was evaluated by MDA assay. (D) The intracellular Fe2+ was assessed in cells described in (B). (E) Lipid ROS levels were detected by flow cytometry after incubation with C11-BODIPY in cells described in (B). (F) Schematic diagram showing a proposed model for the interactions among circRAPGEF5, RBFOX2, and TFRC in EC. CircRAPGEF5 could bind to RBFOX2 protein in the nucleus, where it attenuated the binding of RBFOX2 with pre-mRNA of TFRC and led to the decrease of the labile iron pool and lipid peroxide production, which confers EC cells insensitivity to ferroptosis inducers. Data were mean ± SD. ns, not significant,***P < 0.001 (Student's t-test). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: The effects of CircRAPGEF5 and RBFOX2 on alternative splicing(A) Number and proportion of different alternative splicing events after the circRAPGEF5 knockdown in KLE cells, as identified by the AStalavista tool. (B) Gene Ontology analysis for alternative splicing events related genes. (C) Representative circRAPGEF5 affected skipping exons was validated by using PCR and agarose gel electrophoresis upon knockdown of circRAPGEF5 or RBFOX2 in KLE cells. Data were mean ± SD, **P < 0.01 (Student's t-test).