Mosaic animals in this and subsequent figures were obtained using the MARCM technique (Lee and Luo, 1999) with ey-FLP (Newsome et al., 2000) to induce mitotic recombination in eye imaginal discs. GFP depicts MARCM clones. Posterior is to the right. (A–D’) Wild-type (wt, FRT +) (A), scribble (scrib−/−) (B), RasV12–expressing (C) and scrib−/− RasV12 (D) eye/antennal mosaic imaginal discs from third instar larvae labeled with the ROS indicator Dihydroethidium (DHE). Scale bars: 50 μm. (E) Enlarged scrib−/− RasV12 clones labeled for DHE. Arrowhead in (E’) marks a cell of high DHE labeling. (F) DHE quantification reveals that ROS levels are significantly higher in scrib−/− RasV12 mutant clones compared to wt (FRT +), scrib−/− or RasV12-expressing clones. Plotted is the mean signal intensity ±SD of DHE labelings in clones, analyzed by one-way ANOVA with Holm-Sidak test for multiple comparisons. ****p<0.0001; ns – not significant. Multiple clones from five to ten discs of each genotype were analyzed. (G) Reduction of extra- and intracellular ROS levels in scrib−/− RasV12 mutant clones significantly improves the pupariation rates of animals bearing scrib RasV12 mosaic eye imaginal discs. Expression of UAS-lacZ in scrib−/− RasV12 clones as control has no effect on the pupariation rate. Pupariation rates were determined as the ratio of late stage mutant pupae vs total pupae and were analyzed by one-way ANOVA with Holm-Sidak test for multiple comparisons. Error bars are SD. P values are relative to scrib−/− RasV12 results (left column) and are indicated above the experimental columns. ****p<0.0001; ns – not significant. At least 100 pupae were counted per genotype. Experiments were performed three times. (H–N) Cephalic complexes composed of eye/antennal discs, optic lobes (OL) and ventral nerve cord (VNC) from day 11 old third instar larvae. The genotype is indicated on top of each panel. Expression of UAS-lacZ served as negative control (I). Depletion of ROS strongly reduces clone size (green) and normalizes growth in (J–N). DAPI (blue) labels the outline of the tissue. Scale bars: 200 μm. (O–U) Adult eyes of control (O) and scrib−/− RasV12 mosaics expressing the indicated antioxidant transgenes (Q–U). The percentage number in the top right of each panel indicates the adult survival rate relative to pupal survival. Note that ey-FLP-induced scrib−/− RasV12 MARCM mosaics are 100% lethal (0% adult survival) (P). Genotypes: (A,H,O) yw ey-FLP/+; act>y+>Gal4, UAS-GFP56ST/+; FRT82B tub-Gal80/. FRT82B w+; (B) yw ey-FLP/+; act>y+>Gal4, UAS-GFP56ST/+; FRT82B tub-Gal80/. FRT82B scrib2; (C) yw ey-FLP/+; act>y+>Gal4, UAS-GFP56ST/UAS-RasV12; FRT82B tub-Gal80/FRT82B w+; (D,E,P) yw ey-FLP/+; act>y+>Gal4, UAS-GFP56ST/+; FRT82B tub-Gal80/UAS-RasV12 FRT82B scrib2; (I–N,Q–U) yw ey-FLP/+; act>y+>Gal4, UAS-GFP56ST/UAS-X; FRT82B tub-Gal80/UAS-RasV12 FRT82B scrib2 with UAS-X being UAS-lacZ (I), UAS-DuoxRNAi (J,Q), UAS-hCatS (K,R), UAS-Catalase (L,S), UAS-SOD1 (M,T) and UAS-SOD2 (N,U).