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. 2021 Mar 19;9:627439. doi: 10.3389/fcell.2021.627439

FIGURE 6.

FIGURE 6

The targeting relationships between circ_024949/circ_026259/IGF1 and miR-29b as well as their expression correlations. (A) Sankey plot of the circRNA-miRNA-mRNA network enriched for spermatogenesis-related genes. (B) Putative binding sites revealed by bioinformatics analysis. (C) Dual-luciferase assays were used to validate the binding sites between circ_024949/circ_026259/IGF1-3′UTR and oar-miR-29b. (D) RT-PCR and (E) qPCR analysis for the relative RNA expression. (F) Divergent primers (left panels) and Sanger sequencing (right panels) used for the amplification of circular junctions of circ_024949 and circ_026259. (G) Correlation analysis between oar-miR-29b and circ_024949/circ_026259/IGF1 expression. (H) RNA fluorescence in situ hybridization analysis. (I) IGF1 protein expression assessed by Western blot. (J) Immunofluorescence staining for IGF1 protein. 3M, 1Y, and 3Y refer respectively to 3-month-old, 1-year-old and 3-year-old. Two asterisks (**), one asterisk (*), and ns (non-significant) denote P < 0.01, P < 0.05, and P > 0.05, respectively.