Fig. 4. Circ-4099 and miR-616-5p binding by RIP and RNA–RNA pull-down assays.

a The expression of AGO2 protein can be detected by WB in hNP cells. b RIP assay showed that AGO2 was associated with U1, which acts as a positive control. c, d RIP assay showed that the anti-Ago2 antibody efficiently captured miR-616-5p transcripts; circ-4099 was also detected in these RNA–protein complexes, which shows that it may bind to miR-616-5p transcripts and be indirectly captured by anti-Ago2. e RNA–RNA pull-down qRT-PCR showed an approximately twofold enrichment of circ-4099 in the miR-616-5p-captured fraction compared with the negative control. f WB showed significantly higher AGO2 protein in the miR-616-5p-captured fraction compared with the negative control. g miR-616-5p mimic treatment caused decreased luciferase activity with the LUC-circ-4099 plasmid. h Western blot and i corresponding densitometry analyses showed that Sox9 mRNA expression can be upregulated by circ-4099 over-expression, which can be suppressed by treatment with miR-616-5p mimics. j schematic of constructs Sox9-WT and Sox9-WT containing mutation in miR-616-5p sites generated by site-directed mutagenesis. k Co-transfection with circ-4099 results in a dose-dependent increase in Sox9-WT activity, but no significant induction at the Sox9-MT promoter. l miR-616-5p mimics blocked the inductive effects of circ-4099 on the wild-type Sox9-WT promoter, while no significant responses was observed with the Sox9-MT promoter