Fig. 7
RNA pulldown identifies multiple protein interaction partners of circIGF1R. (A) Filtering strategy to identify protein interaction partners of circIGF1R in non-HF-derived HCFs upon RNA pulldown with circIGF1R-specific probe NC probe (n = 4). (B) Volcano Plot of RNA pulldown experiments from lysates of non-HF-derived HCFs treated with circIGF1R-specific probe or NC probe (n = 4). Dashed lines indicate thresholds (FC ≥ 1.5; padj ≤ 0.05). (C) Venn diagram showing the overlapping proteins between in silico interaction prediction via catRAPID and significantly-enriched proteins. (D) Western Blot of non-HF-derived HCF lysates after RNA-pulldown with circIGF1R-specific probe or NC probe (n = 1). IP: Input; NC: NC probe; circ: circIGF1R probe. (E) Western Blot of HF-derived HCFs treated with mock or circIGF1R mimics including quantified protein levels (n = 3). VCL: Vinculin. (F) BrdU-ELISA in HF-HCFs transfected with either NC siRNA or AZGP1 siRNA, followed by treatment with mock or circIGF1R mimics (n = 3). Data are depicted as fold change and normalized to the cotreatment group receiving mock and NC siRNA. Analyzed via 2-way ANOVA with Sidak’s post-hoc correction. (G) Ki67 immunofluorescence staining in HF HCFs transfected with either NC siRNA or AZGP1 siRNA, followed by treatment with mock or circIGF1R mimics (n = 3). Analyzed via 2-way ANOVA with Sidak’s post-hoc correction. (H) Representative images of Ki67 immunofluorescence staining in HF HCFs transfected with either NC siRNA or AZGP1 siRNA, followed by treatment with mock or circIGF1R mimics. White arrows indicate Ki67+ nuclei. Scale bar = 100 μm.
