Fig. 4: CircPLOD2a/b bind to HuR in U87 cell.

A–D Proteins pulled down by biotin-labeled circPLOD2a (A) or circPLOD2b (C) antisense probes and corresponding scrambled control probes in hypoxic U87 cells were visualized by SDS-PAGE and silver staining. Unique peptides identified by mass spectrometry in (B) circPLOD2a (n = 73) or (D) circPLOD2b (n = 54) compared with corresponding control lanes. E, F Potential proteins interacting with (E) circPLOD2a and (F) circPLOD2b were verified by RNA pull-down assays with corresponding antisense probes in hypoxic U87 cells. Relative integrated density normalized to β-actin was marked above each band. G–J RIP assay confirms the interaction of circPLOD2a/b and linear PLOD2 with HuR. CircPLOD2a/b and linear PLOD2 were detected by RT-PCR (G). The immunoprecipitation products of anti-HuR compared with IgG were treated with or without RNase R. Quantitative enrichment analysis of circPLOD2a (H), circPLOD2b (I) and linear PLOD2 (J) were analyzed by qRT-PCR, n = 3. K Cellular localization of circPLOD2b (red) and HuR (green) were examined by dual RNA-FISH and IF in U87 cells treated with normoxic and hypoxic conditions. Scale bar: 25 μm. L Schematic diagram of wild type and various truncated HuR with Flag-tag. (M) The interaction of circPLOD2a/b with full-length or truncated HuR protein were detected by RIP assay in U87 cells. CircPLOD2a/b were detected by RT-PCR; full-length and truncated HuR proteins were detected by western blot using anti-Flag antibody. N CircPLOD2a and circPLOD2b were immunoprecipitated by anti-Flag beads in U87 cells transfected with plasmids carrying HuR- wildtype (HuR-wt) or truncated forms (HuR-△RRM1/2/3) and detected by qRT-PCR, n = 3. Data are shown as mean ± SEM (error bars) and analyzed using Student’s t-test (H–J) and ANOVA (N).