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.