Cisd2g6528

We obtained the following stocks from the Bloomington Drosophila Stock Center: w¹¹¹⁸ (#3605), UAS-AGBE^IR2 (#42753), Cisd2^G6528 (#30170), Tubulin-Gal4/TM3 Ser.GFP (#5138), UAS-eGFP (#5431), UAS-FLP (#4539), UAS-CD8.Venus (#65609), Vas.Cas9 (#51323). Stocks UAS-AGBE^IR1 (#108087), UAS-IRP1A-RNAi (#105583), UAS-IRP1B-RNAi (#110637), UAS-Cisd2-RNAi (#104501) were obtained from the Vienna Drosophila Resource Center. We used CRISPR/CAS9 to generate the following knock-in and knock-out alleles (see Supplementary Fig. 4 for details): AGBE^FCF, AGBE^FCM, IRP1A^3F, IRP1A^C450S.3F, IRP1A^3R3Q.3F, IRP1B^3F, IRP1A^FCF/TM3 Ser.GFP, IRP1A^KO/TM3 Ser.GFP, IRP1B^KO/KO. We also generated transgenic lines based on the PhiC31 system: UAS-3xFlag-IRP1A^WT, UAS-IRP1A^WT, UAS-3xFlag-IRP1A^C450S, UAS-IRP1A^C450S, UAS-3xFlag-IRP1A^3R3Q, UAS-3xFlag-IRP1B^WT, UAS-IRP1B^WT, UAS-3xFlag-IRP1B^C447S, UAS-IRP1B^C447S, UAS-3xFlag-IRP1B^3R3Q, UAS-Yeast Aco1^WT, UAS-Yeast Aco1^ΔSp, UAS-3xFlag-hIRP1^WT, UAS-3xFlag-hIRP2^WT, dU6-3-IRP1A^gRNA (Supplementary Table 1). y¹w*P[^{nos-PhiC31.NLS;}]X; P[carryP]attP40(II) and y¹w*P[nos-PhiC31/int.NLS]X; P[carryP]attP2(III) were gifts from BestGene Inc. Phm22-Gal4 was a kind gift from Michael O’Connor’s lab. Stocks were maintained on a standard cornmeal diet unless otherwise specified.

Fly husbandry and aging were performed as described (Gargano et al., 2005 (link)). da-Gal4, mef2-Gal4, 188Y-Gal4, appl-Gal4, cisd2^G6528 and all Ppt1 modifiers listed in supplementary material Table S5 were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN). UAS-RNAi transgenic lines for cisd2 (v33925 and v33926), cln3 (v5322) and cln7 (v5089 and v5090) were purchased from the Vienna Drosophila RNAi Center (Vienna, Austria) (Dietzl et al., 2007 (link)). The cisd2 RNAi transgenes do not have predicted off-target effects (defined as genes with at least one continuous stretch of 19 nucleotides complementary to any region of the RNAi transgene (http://stockcenter.vdrc.at/control/vdrcdefinition)). The gmr-Gal4-v33925 and the gmr-Gal4-v33926 double transgenic flies were created by recombining the gmr-Gal4 element and the cisd2 RNAi transgenes onto the same chromosome. UAS-Ppt1 transgenic strains are previously described (Korey and MacDonald, 2003 (link)). Ppt1 loss of function mutant strains (Ppt1^A179T and Ppt1^S77F) were provided by Robert Glaser (Wadsworth Center, Albany NY). The cln3 overexpression strain (UAS-cln3 no. 4) was provided by Richard Tuxworth (Kings College London, London, UK). Sources for all other strains are indicated in supplementary material Tables S3, S4, S5.