Hs gal4

w¹¹¹⁸ flies were used as wild-type control. The w^IR; dcr-2^L811fsX mutant flies have been previously described (27 (link)). ubi-Gal4,tub-Gal80^ts was a gift from Dr. D. Ferrandon. hop^Tum-l, ppl-Gal4, da-Gal4, hs-Gal4 were obtained from Bloomington Stock center. The generation of transgenic UAS-Ect4 and U6:3-gRNA-Ect4 lines was performed as previously described (28 (link)). Ect4-IR was obtained from NIG-FLY stocks (HMJ30091). For the generation of Ect4 mutant lines, transgenic U6:3-gRNA-Ect4 flies were crossed with the nos-Cas9 flies to get male F₀ (nos-Cas9/+; U6:3-gRNA-Ect4/+) that were crossed with w¹¹¹⁸; TM3, Sb/TM6B, Tb to obtain F₁ progenies. Singular F₁ flies were crossed with w¹¹¹⁸; TM3, Sb/TM6B, Tb. PCR products amplified from F₁ flies before being cloned into the pMD19-T vector according to the manufacturer’s instructions (TAKARA) for mutation identification.

The following fly stocks were used in this study: LKB1^X5, UAS-LKB1^WT, and UAS-LKB1^KI [22 (link)], HDAC4^KG09091 (Bloomington #15159), UAS-HDAC4 RNAi (VDRC #20522), UAS-bmm RNAi (Bloomington #25926), UAS-InR^CA (Bloomington #15159), cg-Gal4 (Bloomington #7011), hs-Gal4 (Bloomington #1799), UAS-2xEGFP (Bloomington #6874), UAS-HA-AMPK^TD [12 (link)], CRTC^25-3 [36 (link)], UAS-FLAG-HDAC4^WT and UAS-FLAG-HDAC4^3A [19 (link)], AKHR¹ [7 (link)], and FB-Gal4 [37 (link)]. SIK3^Δ5–31 was generated by imprecise excision of SIK3^G7844 line (KAIST Drosophila Library Facility, Daejeon, Korea). To generate UAS-SIK3 flies, SIK3 EST cDNA (Berkeley Drosophila Genome Project accession no. LD07105) was cloned into the Myc-tagged pUAST vector and microinjected into w¹¹¹⁸ embryos. All flies were grown on food containing approximately 35 g cornmeal, 70 g dextrose, 5 g agar, 50 g dry active yeast (Ottogi, Inc., Korea), 4.6 ml propionic acid, and 7.3 ml Tegosept (100 g/l in ethanol) per liter at 25°C. All flies were backcrossed for a minimum of 6 generations into w¹¹¹⁸ background.

All fly strains and crosses were maintained at 25°C with standard cornmeal medium. w¹¹¹⁸ and yw flies were used as controls. The following alleles were used: For de2f1 mutants, de2f1^rm729 [22 (link)] alleles were crossed to the Df(3R)Exel6186 deficiency allele which lacks the entire de2f1 gene locus (Exelis collection at the Harvard Medical School). dDP^a3a1 and dDP^a4a3 alleles [7 (link)] were crossed together to generate dDP mutants. PiggyBac transposase stock (#8285) was obtained from the Bloomington Stock Center for removal of the ScarlessDsRed cassette for de2f1b mutant generation. For overexpression and rescue experiments, the following GAL4 lines were obtained from the Bloomington Drosophila Stock Center: Ubi-Gal4, GMR-Gal4, and hs-Gal4 (Bloomington, IN, USA). For knock-down of CycE, UAS-CycE-RNAi was obtained from the Vienna Drosophila Resource Center (Vienna, Australia). PCNA-GFP was obtained from Dr. Duronio [27 (link)]. UAS-FM-dE2F1a and UAS-FM-dE2F1b overexpression constructs were generated by using the Drosophila Gateway collection (Drosophila Genomic Resource Center). The entry clones, pENTR-dE2F1 and pENTR-dE2F1b, were generated then recombined into the pTFM destination vector to be randomly integrated into the Drosophila genome. Minimum of 10 independent transgene lines were screened to identify lines with similar levels of expression.

Drosophila lines used in the experiments were hs-GAL4 (2077; the Bloomington Drosophila Stock Center), mef2-GAL4 (27390; the Bloomington Drosophila Stock Center), PINK1^B9 (34749; the Bloomington Drosophila Stock Center), park¹ (34747; the Bloomington Drosophila Stock Center), UAS-CISD RNAi (33925 and 104501; the Vienna Drosophila Resource Center), UAS-Itpr (30742; the Bloomington Drosophila Stock Center), UAS-Itpr RNAi (6484; the Vienna Drosophila Resource Center), UAS-ERGCaMP6-210 (83294; the Bloomington Drosophila Stock Center), UAS-GCaMP5G (42037; the Bloomington Drosophila Stock Center), UAS-Luciferase RNAi (31603; the Bloomington Drosophila Stock Center), and UAS-DsRed (6282; the Bloomington Drosophila Stock Center). The UAS-CISD WT-HA was generated by microinjecting pUAST-CISD-HA into w¹¹¹⁸ embryos. The RNAi lines for Parkin substrate genes used in Fig. 2a were described in Supplementary Table 1. All Drosophila stocks were maintained at 25 °C on the standard cornmeal-yeast-agar medium.