Figure 8.

Upon autophagy inhibition, ROS accumulate in Ras-activated tissue and lead to cooperative tissue overgrowth via JNK activation. (a) Oxidative stress levels in eye-antennal mosaic discs as detected by CellROX Deep Red staining. Autophagy inhibition using Atg8a^RNAi did not augment ROS levels compared with control discs. Ras activation (Ras^V12 GFP) slightly increased ROS levels. However, massive accumulation of ROS was detected in Ras^V12Atg8a^RNAi mosaic tissue. Interestingly, ROS are not limited to Ras^V12Atg8a^RNAi clones, but spread in the whole disc, suggesting a non-cell-autonomous regulation of oxidative stress levels in Ras^V12Atg^RNAi mosaic tissue. Scale bar: 50 μm. (b) Quantification of ROS levels in whole discs from (a). (c) ROS levels in Ras^V12 Atg8a^RNAi mosaic discs are reduced upon SOD1 overexpression, but not upon JNK blockage, confirming that JNK activation is downstream of ROS accumulation in Ras^V12 Atg8a^RNAi discs. Note that overexpression of SOD1 rescues both cell-autonomous and non-cell-autonomous accumulation of ROS, and restores the size and shape of Ras^V12 Atg8a^RNAi eye discs. (d) Quantification of ROS levels in whole discs from (c). (e) Scanning electron micrographs of adult eyes expressing Ras^V12 and GFP (first column), Atg13^RNAi (second column), Atg8a^RNAi (third column) or Syx17^RNAi (fourth column), in combination with control lacZ (first row), bsk^DN (second row) or SOD1 (third row). Ras^V12Atg^RNAi eye overgrowth is rescued by overexpression of bsk^DN or SOD1, showing that autophagy-impaired Ras^V12-driven tumourigenesis is due to elevated ROS levels and JNK signalling. Note that overexpression of SOD1 and bsk^DN also rescues Ras overgrowth alone (first column). Error bars=s.e.m. Statistics: one-way ANOVA with Tukey's multiple correction.