Rescue of Fertility and Pupal Lethality in Drosophila

Methoprene (Service Chemical Inc., Germany), JH III (Sigma-Aldrich), and MF (Echelon) were purchased. JHB3 was synthesized from MF using m-chloroperbenzoic acid in dichloromethane (Sigma-Aldrich) [19 (link)]. For rescue of fertility of jhamt², newly eclosed females were placed in vials with standard medium; after 24 hours, virgin females were topically treated with acetone-dissolved methoprene (0.5 μl × 10^-3 M per female) [21 (link), 23 (link)]. For rescue of pupal lethality of Aug21>grim and jhamt²hmgcrRNAi, methoprene, JHB3, JH III, and MF (0.5 μl × 10^-9~-2 M per larva) were dissolved in acetone and topically applied to the larvae at 96h AIW [14 (link), 20 (link), 21 (link), 31 (link), 33 ]. For inducing Kr-h1 expression in w¹¹¹⁸ and Met²⁷gce^2.5k, fat body tissues were isolated at 96h AIW and treated with methoprene, JHB3, JH III, and MF (1×10^-6 M; DMSO as a control) for 30 min. For testing the conversion of MF to other JHs, the jhamt²hmgcrRNAi larvae were topically treated with acetone or MF (0.5×10^-2 μmol per larva) at 108h AEL, and the three sesquiterpenoids titers were measured at 3hAIW (about 24 hours after treatment).
For inducing Kr-h1 expression in Drosophila Kc cells cultured in Schneider’s medium, the cells were treated with methoprene, JHB3, JH III, and MF (1×10^-11~-6 M; DMSO as a control) for 30 min [31 (link)]. Using the T7 RiboMAX Express RNAi System (Promega), dsRNAs of USP and EGFP (as a control) were synthesized. Reduction of gene expression by RNAi in Kc cells was performed by transfecting dsRNAs using Effectene at a final concentration of 1 μg/ml dsRNA. The transfected cells were cultured for 48 h and treated with MF (1×10^-6 M; DMSO as a control) for 30 min [31 (link)].