Fig. 2

CircLIFR binds to MSH2 protein. a Biotin-labeled sense or antisense circLIFR probes were used for RNA-protein pull-down against T24 cell lysates. Identification of proteins that interact with circLIFR by silver staining. Red arrow indicates the major differential band precipitated in T24 lysates. b Analysis pipeline was performed to identify proteins that interact with circLIFR: (1) The 149 proteins that were only pulled down by sense probe were screened; (2) The 15 proteins with molecular masses of 100–130 kDa were then selected as the candidates according to the positive band found in silver staining; (3) MSH2 was selected as it was the only protein with high abundance (no less than 3 peptides). c Mass spectrometry assay depicted the MSH2 peptides pulled down by sense circLIFR probes. d MSH2 immunoblot analysis of the biotin-labeled sense and antisense circLIFR probes pull-down eluate from lysates of T24 and UMUC3 cells. GAPDH was used as loading control. e RNA immunoprecipitation (RIP) assays in T24 and UMUC3 cells using MSH2 and IgG antibody. The precipitate was subjected to western blotting with the antibodies against MSH2 and GAPDH. The MSH2-enriched circLIFR relative to the IgG-enriched value was calculated by qRT-PCR. Data were mean ± SD. *P < 0.05, **P < 0.01 (Student’s t-test). f Dual RNA-FISH and immunofluorescence staining assay indicating the co-localization of circLIFR (red) and MSH2 (green), with nuclei staining with DAPI (blue). g Prediction of circLIFR-MSH2 interaction by using the catPAPID algorithm and schematic of MSH2 with functional protein domains. MSH2 truncations lacking the region 620–934 aa (3xFlag Δ620–934), 300–934 aa (3xFlag Δ300–934), 1–619 aa (3xFlag Δ1–619), or 1–299 aa (3xFlag Δ1–299). h Relative enrichment of endogenous circLIFR in truncated MSH2 RIP was measured by qRT-PCR, following T24 cells transfected with 3xFlag-MSH2 truncations. Data were mean ± SD. ns, not significant, **P < 0.01 (Student’s t-test)