Fig. 5: IGF2BP1 associated with mRNAs that encode essential regulators of spermatogonial stem cell (SSC) maintenance.
From: RNA-binding protein IGF2BP1 is required for spermatogenesis in an age-dependent manner
a Immunoblot analysis of IGF2BP1 immunoprecipitation using cultured germline stem cells (GSCs). b Venn diagram showing the overlapping transcripts identified by RNA immunoprecipitation-sequencing (RIP-Seq) of GSCs and downregulated differentially expressed genes (DEGs) in the conditional knock-out (cKO) A1 spermatogonia cluster by single cell RNA-sequencing (scRNA-Seq). c Gene Ontology enrichment analysis of the overlapping transcripts identified by RIP-Seq of GSCs and downregulated DEGs in the cKO A1 cluster by scRNA-Seq. d, e RIP-qPCR analysis of IGF2BP1 (d) and ELAVL1 (e) potential targets in P10 mouse testes, with Gapdh as a negative control. Relative enrichment in IGF2BP1-IP and ELAVL1-IP was compared with input (n = 3). Bar graphs represent the means ± standard deviations. ns, not significant, ****P < 0.0001. f Luciferase reporter analysis demonstrating reduced activity of indicated reporters in Igf2bp1-knockdown (shRNA, red) compared with control HEK293T cells (shNC, blue). ns, not significant, ***P < 0.001. g Reducing LIN28A mRNA half-life by silencing IGF2BP1 in Huh-6 cells. Bar graphs represent the means ± standard deviations. h Western blotting assay to compare the protein expressions of LIN28A between 8-month-old wildtype (WT) and Igf2bp1 cKO mice. i The band intensity of LIN28A was normalized to GAPDH. ***P < 0.001. j, k Immunostaining of LIN28A in testes from 8-month-old WT and Igf2bp1 cKO mice (mice n = 3; tubules per mouse, n = 40). Cell nuclei were stained with DAPI. The white arrows indicate LIN28A-positive cells. Fluorescence intensity of LIN28A was normalized to DAPI intensity. Bar graphs represent the means ± standard deviations. **P < 0.01.
