Drug–target validation. (A) Steady-state expression levels of sGFP::ATZ in the N2, age-1(hx546) and daf-16(m26) backgrounds. Data are normalized to N2;sGFP::ATZ worms. (B–D) Effect of wortmannin on steady-state levels of sGFP::ATZ. N2;sGFP::ATZ (B), sGFP::ATZ;age-1(hx546) (C) and sGFP::ATZ;daf-16(m26) (D) animals were treated with wortmannin (100 µm) for 24 h and analyzed using the ArrayscanVTI. GFP(RNAi) treatment was included as a control to show that ATZ levels could be further reduced in each line. Note wortmannin reduced the sGFP::ATZ level in the wild-type N2 but not in age-1(hx546) or daf-16(m26) mutant backgrounds. (E) Effect of various drugs on sGFP::ATZ;daf-16(m26) animals. Of the drugs known to decrease sGFP::ATZ levels in the N2 background, only wortmannin failed to reduce sGFP::ATZ in the daf-16(m26) background. (F) ATZ::GFP animals were treated with 5, 12.5 or 50 μm of fluphenazine and wortmannin, either singly or in combination. The data were normalized to the untreated DMSO control within each experiment. All experiments were repeated at least three times with n > 150 animals/treatment. Error bars represent SD (A–E) or SEM (F). Statistical significance was determined using the Student's t-test. ***P < 0.001, **P < 0.01, *P < 0.05.