Fly stocks and generation of transgenic flies

All stocks were cultured in standard medium at 25°C. The w1118 strain was used as the WT control in this study. The Drosophila Unr hypomorphic mutant line w1118; PBac{PB}Unrc01923 (FlyBase ID: FBst0010757) was previously mapped and tested (50) and was used as a mutant (dunr) line in this study. The deficiency strains that removed Unr, Df(3L)BSC815 (abbreviated as Df1) and Df(3L)BSC157 (abbreviated as Df2), were obtained from the Bloomington Stock Center (catalog nos. 27576 and 9544, respectively). UAS-dUnr-RNAi was obtained from Tsinghua Fly Center with the genotype y1sc*v1; P{TRiP.HMS00494}attP2/TM3, Sb1 (catalog no. THU0937). The whole body–expressed Da-Gal4 and pan-neuron–expressed Elav-Gal4 were supplied by Z. Zhang (Center South University, Changsha, Hunan Province, China). Western blotting with rabbit anti-dUnr antibodies was performed to identify the expression level of dUnr in dunr, dunr/Df1, dunr/Df2, and Da-RNAi lines by extracting protein from the whole body of the 4-day-old male flies. Antibody to dUnr was provided from Fatima Gebauer Lab (Centre de Regulacio Genomica, 08003 Barcelona, Spain) (51) and used at a dilution of 1:500. Anti–β-actin (A2228, Sigma-Aldrich; 1:5000) was used as loading control. UAS-dUnr and UAS-hCSDE1 transgenic flies were generated by embryo injection into w1118 with a recombinant pUAST vector containing the full-length dUnr or hCSDE1 protein-coding sequence, respectively. To generate the dUnr-pUAST vector, the coding sequence for full-length Drosophila Unr (dUnr, NM_001300054.1) was obtained by PCR amplification from cDNA and synonymously mutated for resistance to the RNAi fragment of UAS-dUnr-RNAi and then subcloned into the pUAST with a Flag tag on the N terminus. The full-length human CSDE1 coding sequence (hCSDE1, NM_001242891, GeneCopoeia) was subcloned into the pUAST with the same tag. Genomic DNA PCR and Western blotting with anti-Flag antibody were performed to identify these transgenic lines. Elav-Gal4/+; UAS-dUnr/+; UAS-dUnr-RNAi and Elav-Gal4/+; UAS-hCSDE1/+; UAS-dUnr-RNAi flies were used as dUnr and hCSDE1 rescue lines. Study protocols comply with all relevant ethical regulations and were approved by the IRB of Central South University.

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