Research Paper Volume 11, Issue 1 pp 185—208

Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis

Figure 6. Chronic rapamycin treatment decreases piRNAs in mouse testes. (a) Reduction of piRNAs in isolated pachytene spermatocytes and round spermatids from adult rapamycin-treated mice. Levels of 18S and 28S ribosomal RNAs were used as a loading control. (b) Immunoprecipitation (IP) of MILI-associated piRNAs from control and rapamycin-treated testis followed by 32P-end-labeling. MILI protein levels were determined by western blotting. (c) MILI-IP complexes from control testes lysate in (b) were diluted (1:2). (d, e) Length distribution in total small RNA libraries generated from isolated pachytene spermatocytes (d) and round spermatids (e). (f) Levels of cluster-derived small RNA reads (24-32nt) in rapamycin-treated relative to control round spermatids. Reads were mapped to pachytene piRNA clusters and the top 30 piRNA clusters producing the largest amounts of piRNAs are shown.