Fig. 7.
Feeding ATP to larvae depleted for some nucleotide synthesis enzymes rescued their AO phenotype. (A) Graph showing that depletion of ADSS, PRPS2 or JNK in CIN cells (induced by mad2RNAi) showed high AO staining, which was rescued by feeding the larvae with rATP (1 mM). No rescue was observed in ATP fed larvae of genotype TKLRNAi mad2RNAi. Male larvae with high CIN induced by Rad21 depletion (UAS-rad21RNAi, UAS-Dicer2) also showed high AO staining that could be rescued by rATP feeding, while feeding ATP to female larvae of the same genotype did not show reduction in their AO phenotype. Candidate knockdowns in CIN cells are represented by black bars and rATP fed candidate knockdowns in CIN cells are represented by grey bars. The error bars represent the 95% CIs, n≥10 in all cases. The experiment was repeated three times. Representative images are shown in Fig. S6. (B) Feeding the larvae with dATP (1 mM) rescued the AO phenotype in ADSS and PRPS2 knockdowns in CIN cells (grey bars) as compared to their controls (black bars). No rescue was observed in dATP fed larvae of genotypes TKLRNAimad2RNAi and in high-CIN larvae (UAS-rad21RNAi, UAS-Dicer2). The error bars represent the 95% CIs, n≥10 in all cases. The P-values were calculated by two-tailed t-tests with Welch's correction: ****P<0.0001, **P<0.01, *P<0.05; ns, non-significant. This experiment was repeated twice. Representative images are shown in Fig. S5. (C) Graph showing Quinacrine (QA, a marker for lysosomal ATP) staining on larval wing discs. High QA staining was observed, when nucleotide synthesis candidates were depleted in CIN cells (dark grey bar) as compared to candidates knocked down in non-CIN cells (light grey bar). Wing discs were incubated with bafilomycin (150 nM) for 30 min and then stained with QA (15 uM). Candidates' knockdown in CIN cells (black bar) show significantly lower QA staining after bafilomycin treatment. Quantifications show the normalized grey value of staining, which is obtained by subtracting the mean grey value of wild type from the affected region of each disc. Error bars indicate 95% CIs, n≥10 in all cases. The P-values were calculated by two-tailed t-test with Welch's correction: ****P<0.0001, ***P<0.001, **P<0.01; ns, non-significant. Representative images are shown in Fig. S6. (D) Graph showing that blocking v-ATPase by bafilomycin (75 nM) inhibits the AO phenotype in nucleotide candidate knockdowns in CIN cells. When wing discs of candidate knockdowns in CIN cells were incubated with bafilomycin for 30 min it significantly reduced the AO staining in these discs compared to controls. Quantifications show the normalized grey value of staining, obtained by subtracting the mean grey value of wild type from the mean of the affected region of each disc. The error bars represent 95% CIs, n≥12 in all cases. The P-values were calculated by two-tailed t-tests with Welch's correction. This experiment was repeated twice. Representative images are shown in Fig. S6.