Actin5c gal4

Fly stocks and genetic crosses were reared at 25°C unless otherwise stated. Fly stocks were kept in vials or bottles containing standard fly food (0.8% Drosophila agar, 5.8% Cornmeal, 5.1% Dextrose, and 2.4% Brewer’s yeast). The following fly strains were used in this study: insc-Gal4 (BDSC#8751; 1407-Gal4), insc-Gal4, UAS-Dicer2 with and without UAS-CD8-GFP, Jupiter-GFP (G147), UAS-hyx, UAS-HRPT2 [51 (link)]. The type II NSC driver: w; UAS-Dicer 2, wor-Gal4, ase-Gal80/CyO; UAS-mCD8-GFP/TM3, Ser [86 (link)]; hyx RNAi hyx^HT622, UAS-venus-polo [28 (link)].
The following stocks were obtained from Bloomington Drosophila Stock Center (BDSC): UAS-Gal RNAi (BDSC#50680; this stock is often used as a control UAS element to balance the total number of UAS elements), ctr9^12P023 (BDSC#59389), rtf1 RNAi (BDSC#36586), rtf1 RNAi (BDSC#34850) [87 (link)], rtf1 RNAi (BDSC#31718), UAS-aurA (BDSC#8376), actin5C-Gal4 (BDSC#25374).
The following stocks were obtained from Vienna Drosophila Resource Center (VDRC): hyx RNAi (28318), hyx RNAi (103555), atms RNAi (108826) [87 (link)], atu RNAi (17490) [88 (link)], atu RNAi (106074), ctr9 RNAi (108874), ctr9 RNAi [89 (link)], rtf1 RNAi (27341) [88 (link)], rtf1 RNAi (110392).
All experiments were carried out at 25°C, except for RNAi knockdown or overexpression experiments that were performed at 29°C.

Fly stocks were maintained on cornmeal/agar/yeast food at 21°C, except where noted. Before immunofluorescence staining, newly eclosed flies were fed wet yeast paste every day for 2–4 days. Unless specified, yw (BDSC 1495) was used as the control. The following stocks were obtained from the Bloomington Drosophila Stock Center: c355 GAL4 (BDSC 3750), actin-5C GAL4 (BDSC 8807), mgst1^KG04713 (BDSC 13839), Su(P)^EY13245 (BDSC 20866), p23^EY05607(BDSC 16661), akr1B^PL00034 (BDSC 19594), akr1B^EY07011 (BDSC 16777), p23 RNAi HMJ24151 (BDSC 62911), p23 RNAi-2 GL01292 (BDSC 41862), akr1B deficiency-1 DF(3L)BSC577 (BDSC 25411), akr1B deficiency-2 DF(3L)ED4475 (BDSC 8069), akr1B RNAi HMS05657 (BDSC 67838), akr1B RNAi-2 HMC05226 (BDSC 62219) and UAS Dicer 2 (BDSC 24651). The following stocks were obtained from the Exelexis Stock Center: mgst1^d10243, akr1B^d00405 and pxt^f01000 (Thibault et al., 2004 (link)). The oskar GAL4 line (second chromosome; BDSC 44241) was a generous gift from Anne Ephrussi [European Molecular Biology Laboratory; (Telley et al., 2012 (link))]. Expression of the RNAi lines were achieved by crossing to actin-5C GAL4, c355 GAL4 or oskar GAL4, maintaining fly crosses at 21°C and maintaining progeny at 29°C for 5–6 days. UAS Dicer was used in combination with c355 to enhance RNAi efficiency where noted in the figure legends.

RNAi lines used in this study were procured from the Vienna Drosophila Resource Centre (VDRC) (Dietzl et al. 2007 (link)). Flies were cultured at 25° on a corn meal-agar medium containing yeast granules. A bipartite UAS/Gal4 system (Brand and Perrimon 1993 (link)) was used for tissue-specific knockdown of chaperones. The Gal4 driver lines used in this study were actin5C-Gal4 (BDSC-25374), ey-Gal4 (BDSC-5534), D42-Gal4 (BDSC-8816), and elav^C155-Gal4 (BDSC-458). The white-eyed w¹¹¹⁸ Drosophila strain was used as control except where indicated. All the RNAi knockdown experiments were performed at 29° under controlled humidity (60% RH) in an incubator. To screen for essential chaperones, actin5C-Gal4 without Dicer-2 was used. While Dicer-2 does enhance the efficiency of knockdown, it may also cause off-target knockdowns and lead to additional lethality (Dietzl et al. 2007 (link)). Hence, we chose not to use Dicer-2 for screening chaperones.