Figure 6. OSU-03012 alters the nuclear localization and the activity of DAF-16.

(A) Nuclear accumulation of DAF-16 induced by OSU-03012 treatment. Images of TJ356 animals, which carry an integrated daf-16::gfp array in a wild-type background, exposed to either DMSO control or 0.5 µM of OSU-03012 for 72 hours. (B) Quantification of the nuclear accumulation of DAF-16::GFP in response to 10 µM celecoxib and 0.5 µM OSU-03012 treatments. Worms were scored for the presence or absence of GFP accumulation within the intestinal nuclei at the first day of adulthood (n ≥ 120 for each treatment). An animal was scored as having nuclear DAF-16 if more than one intestinal nucleus contained DAF-16-GFP. RNAi treatment by feeding of daf-2 was also performed as a positive control. Lifespans following each drug treatment were determined to confirm the effectiveness of the drug treatment. (C) Effects of celecoxib and OSU-03012 on DAF-16 transcriptional activity. Wild-type N2 animals were exposed to DMSO control, 10 µM celecoxib, or 0.5 µM OSU-03012 from hatching. Relative mRNA levels of sod-3 of these animals were measured by quantitative RT-PCR and the mean of three different sample sets are shown. The relative mRNA levels were normalized against act-1 (beta-actin) levels. Error bars: ± STD. (D) daf-2(e1370) mutants (P₀) were exposed to DMSO control or 0.5 µM OSU-03012 at 20°C. The F₁ eggs were then moved to the different temperatures indicated for 72 hours before being scored for dauer formation. Each bar represents combined data of three independent experiments per condition (n ≥ 150 for each treatment). Asterisks indicate significant changes (*, P < 0.005). P values were calculated by Pearson's chi-square test.