Figure 7.

Knockdown of RNase X25 protects Drosophila from oxidative stress. (a) Cohorts of six female control (open bars, w¹¹¹⁸) and RNase X25 fat body knockdown (filled bars, CG8194) flies were weighed. Body weight is reported as mean weight per fly+S.E. for n=3 independent crosses. (b–e) Flies of each genotype were fed 5% sucrose (−) or 5% sucrose with 15 mM paraquat (+) for 20 h. Total RNA was isolated from fat body-enriched tissue of eight flies, oligo dT primed for cDNA synthesis, and expression of RNase X25, moi, and tgs1 was determined by qRT-PCR relative to tubulin (b, graph shows mean+S.E. for n=3 independent crosses). Mean (+S.E.) survival at 20 h was quantified for 60 flies per genotype/condition in n=3 independent crosses (c). Extracts were prepared from fat body-enriched tissue pooled from eight flies. Carbonylated proteins were derivitized using DNPH or control solution, and analyzed by western blotting using α-DNP antibody. Representative blot is shown (d, left) and graph (d, right) quantifies results from four independent crosses. 7-keto cholesterol was quantified in fat body lipid extracts by LC-MS/MS and is reported normalized to protein concentration (e, mean+S.E. for n=3 crosses). *P<0.05 for comparisons indicated