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. 2011 Sep 16;30(21):4465–4478. doi: 10.1038/emboj.2011.318

Figure 6.

Figure 6

BI-1 controls autophagy activation in vivo in Drosophila melanogaster. (A) The expression of dBI-1 was knocked down in Drosophila melanogaster. Then, relative expression levels of dBI-1 mRNA were monitored by semiquantitative PCR. Actin levels were monitored as control. (B) LC3 levels were monitored in control (Da-Gal4>huLC3:GFP) or dBI-1 RNAi larvae (Da-Gal4>huLC3:GFP, Dcr2, dBI-1i) under basal or fasting conditions. Then, huLC3–GFP levels were analysed by western blot. In addition, dBI-1i larvae were treated with 100 μM SP600125 (added in the growing media). (C) Left panel: the presence of LC3-positive vesicles (white arrowheads) was monitored by confocal microscopy in control (Da-Gal4>huLC3>GFP) and dBI-1 knockdown pupae (Da-Gal4>huLC3:GFP, Dcr2, dBI-1i) after 6 h of puparium formation. The organization of actin cytoskeleton was monitored by staining with phalloidin (Ph, red). Nucleus was stained with Topro (blue). Scale bar: left 20 μm and right 11 μm. Right panel: overexpression of dBI-1 (dBI-1EY03662) delays salivary gland degradation. Overexpression of dBI-1 was confirmed by semiquantitative RT–PCR. Actin levels were monitored as loading control. Right panel: wild-type control and dBI-1EY03662 pupae were analysed at 14 h after puparium formation. Superficial and internal confocal planes of the cells are presented. Scale bar: 40 μm. (D) Wild-type control and dBI-1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker (red) and Hoechst (blue). Then, lysosomal content in the fat body was visualized by epifluorescence microscopy. The percentage of cells presenting lysotracker-positive stain is indicated. Scale bar: 50 μm. (E) Control or dBI-1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days. In all, 100 individuals were monitored in each condition. Data represent mean and standard error (N=3). Two-way ANOVA was used to analyse statistical significance between groups. (F) Second instar dBI-1 RNAi or control larvae were grown in food supplemented with 25 μg/ml Tm dissolved in DMSO or 0.5% DMSO as control. The number of individual reaching the adult fly stage was evaluated. Mean and standard error are presented (N=3), **P<0.01.