Figure 3.

Characterization of the small chaperones and autophagy. (a) Transcription level of Hsp26 and Hsp27 in the CG17259 mutant detected by qRT-PCR. The RNAs were collected from the fly heads. The relative mRNA level of Hsp26, Hsp27, Hsp70 and Hsp83 in the wild type fly (w ¹¹¹⁸) was defined as 1. The means ± SD of relative transcripts. Trial n = 3. (b) The Western blot to detect the protein level of Hsp26 and Hsp27 in the CG17259 mutant flies. The heterozygous mutants of hsp26 and hsp27 serve as controls to indicate the specificity of the antibodies. Trial n = 3. (c) qPCR for Hsp26 and Hsp27. The heterozygous mutant of Hsp26^−/+ and Hsp27^−/+ lines contain a third chromosome balancer (short bristle) and they are homozygous lethal. The relative levels of transcript of Hsp26 and Hsp27 to the wild type (w ¹¹¹⁸) are shown. Trial n = 3. (d) Survival of AG flies under the genetic background indicated. Trial n = 6. (e) Survival of AG flies under the genetic background indicated. Trial n = 5. (f) Autophagy activation determined by the in vivo reporter (mCherry-Atg8a, the red channel). In the fed condition, the fat bodies of wild type (w ¹¹¹⁸) showed minimum activation of autophagy, but autophagy was activated under the starvation condition. This response serves as a positive control for autophagy activation. In the CG17259 mutant flies, autophagy was activated under the fed condition. Trial n = 2. (g) Survival of AG flies under different autophagy mutant backgrounds. The RNAi and mutant of the Atg genes (as indicated as LOF) all showed enhancing effect on lethality of AG flies; whereas up-regulation of several Atg genes (GOF) all showed a rescue effect on lethality of AG flies. Trial n = 5.