Inhibition of Hsp90 reduces Siwi and BmAgo3 proteins in vivo. (A) Wild-type (WT) BmN4 cells, Flag-Siwi-expressing cells, and Flag-BmAgo3 expressing cells were treated with DMSO or 10 μM 17-AAG for 48 h. Total cell lysates were subjected to Western blot analysis with anti-Siwi, anti-BmAgo3, anti-Flag, and anti-Actin antibodies. Actin served as a loading control. (B) The levels of Siwi and BmAgo3 mRNAs in the BmN4 cells treated with DMSO or 10 μM 17-AAG for 48 h were measured by qPCR. rp49 served as an internal control. The average ± standard deviations from three independent trials are shown. (C,D) Wild-type (WT) BmN4 cells, Flag-Siwi, or Flag-BmAgo3 stably expressing cells were treated with DMSO or 10 μM 17-AAG for 48 h. The cell lysates were subjected to immunoprecipitation with anti-Flag antibodies and analyzed by Western blot (C). RNAs bound to Flag-Siwi or Flag-BmAgo3 were purified from the immunoprecipitates and detected by the 3′-end radiolabeling with [α-32P]cordycepin 5′-triphosphate (D). Siwi- and BmAgo3-bound piRNAs were reduced in proportion to the reduction of the Piwi proteins. (*) An ∼16-nt short RNA accumulation in BmAgo3 immunoprecipitates in the presence of 17-AAG.
