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Open Access Research article

Drosophila larvae lacking the bcl-2 gene, buffy, are sensitive to nutrient stress, maintain increased basal target of rapamycin (Tor) signaling and exhibit characteristics of altered basal energy metabolism

Jessica P Monserrate, Michelle Y-Y Chen and Carrie Baker Brachmann*

Author Affiliations

Developmental and Cell Biology, University of California, Irvine, CA 92697, USA

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BMC Biology 2012, 10:63  doi:10.1186/1741-7007-10-63

Published: 24 July 2012

Additional files

Additional file 1:

Figure S1. (A) Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) demonstrating knockdown of buffy transcript levels in two lines in which buffy RNAi was expressed using the ubiquitous driver daughterless-Gal4. (B-D) Fat body Nile Red stain of RNAi lines reared in restrictive media (20% cornmeal/yeast/agar food (CY); 1.8% sucrose, compare to Figure 1D) phenocopies observations made in the buffyH37 null mutant. (B) da-Gal4 driver alone; (C) da-Gal4, UAS-buffyRNAi498; (D) da-Gal4, UAS-buffyRNAi499. (E-G) LysoTracker Red (LTR) stain on fat bodies from RNAi lines amino-acid starved for 2 h also phenocopies the buffyH37 null mutant. (E) da-Gal4 driver alone; (F) da-Gal4, UAS-buffyRNAi498; (G) da-Gal4, UAS-buffyRNAi499 (compare to Figure 4A).

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Additional file 2:

Figure S2. Immunoblot using phosphospecific S6K antibody conducted on larval lysates from buffy knockdown lines. RNAi line 499 reproduced the increase in phosphorylated S6K that is observed in the buffy null allele.

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Additional file 3:

Figure S3. (A) Quantification of LysoTracker Red (LTR) stain from three different wild-type alleles to determine variation in autophagic responses to 2 h of starvation. Graph represents numbers from 11 different animals. The buffy mutant is included for comparison. (B) LC3-green fluorescent protein (GFP) marker highlights autophagic vacuoles to corroborate LTR data. Shown are fat bodies from 2 h-starved larvae expressing LC3-GFP (green, middle panels) and stained with LTR (red, top panels) with merged images shown in the bottom panels. More LC3-GFP punctae are observed in the buffyH37 mutant and wild-type larvae starved for 2 h. Yellow arrowheads point to examples of LC3-GFP punctae that colocalize with LTR (insets in wild-type images), indicating autolysosomes. Genotypes for (B): cg-Gal4, UAS-LC3-GFP and cg-Gal4, UAS-LC3-GFP, buffyH37.

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Additional file 4:

Figure S4. LysoTracker Red (LTR) stain (red in left panels, grayscale in right panels) of single cell clones overexpressing dominant negative Tor (TorTED, green) in the buffyH37 mutant and wild-type background. Clones of TorTED in wild-type fat body (A) or buffyH37 fat body (B) from fed larvae are autophagic and small as expected. Similarly, clones of TorTED in wild-type fat body (C) or buffyH37 fat body (D) from larvae starved for 2 h are indistinguishable in their autophagic response. Note that in both cases, TorTED cells have the same amount of autophagy as neighboring Tor+ cells in the starved conditions. The TorTED clones observed in (C) and (D) were generated later in development as opposed to those observed in (A) and (B), which accounts for the difference is cell size. Images are representative of 7 to 10 different animals surveyed per genotype. Genotypes: (D, F) hs flp/+; UAS-TorTED/+; Act > CD2 > Gal4, UAS-GFP/+. (E, G) hs flp/+; buffyH37 , UAS-TorTED/buffyH37; Act > CD2 > Gal4, UAS-GFP/+.

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