Fig. 5
circBIRC6 direct interaction with XRCC4 enhances its chromatin localization. (A) Silver staining of RNA pull-down assay revealing the distinct band associated with the biotin-labeled circBIRC6 probe in Panc-1 cells treated with CAF-derived EVs. (B-D) Mass spectrometry (B-C) and western blot (D) analysis identify XRCC4 as a potential circBIRC6 interacting protein. (E) RIP assay validating circBIRC6’s interaction with XRCC4 in Panc-1 cells treated with CAF-derived EVs. (F-G) mRNA and protein levels of XRCC4 in Panc-1 cells post-treatment with EVs from CAFs transfected with lenti-circBIRC6-shRNA or lenti-NC-shRNA. n.s., not significant. (H) Western blot examination of the cytoplasmic and nuclear distribution of XRCC4 in Panc-1 cells treated with EVs from CAFs transfected with lenti-circBIRC6-shRNA or lenti-NC-shRNA. (I-J) In vitro RNA/protein interaction assay between biotin-labeled circBIRC6 fragments and recombinant XRCC4 protein. (K) RNAalifold-based prediction of circBIRC6 secondary structure. The prediction was based on minimum free energy (MFE). Color scale indicates prediction confidence. Red shades reflect strong prediction confidence. (L) The binding of wild-type or mutated circBIRC6 to XRCC4 was evaluated by RNA immunoprecipitation (RIP) in Panc-1 cells. n.s., not significant. (M) The subcellular distribution of XRCC4 in the cytoplasm and nucleus of Panc-1 cells transfected with wild-type or mutated circBIRC6 was examined using Western blot analysis. Data are expressed as mean ± SD. **p < 0.01