2.15 Analysis for DAF-16 nuclear localization

To observe subcellular localization of DAF-16 transcription factor, TJ356 [daf-16p::daf-16a/b::GFP + rol-6(su1006)] in which GFP-tagged DAF-16 protein were used in this assay. Synchronized L3 larvae were exposed to 6-OHDA and supplemented with different doses of HLEA-P1 as described previously. After 72 h of treatment, worms were washed three times with M9 buffer and transferred to 2% agarose pad on a glass slide for fluorescence imaging under a fluorescence microscope (BX53; Olympus Corp., Tokyo, Japan). Subcellular localization of DAF-16 was categorized as nuclear, intermediate and cytosolic. In this study, worms exhibited strong nuclear fluorescence dots were counted as nuclear. Worms having no fluorescence dot in cytoplasm were counted as cytosolic, while worms exhibited unclear cytosolic DAF-16 and unclear nuclear fluorescence dots were assigned as intermediate. All conditions were performed in three independent replicates. In each replicate, approximately 30 worms were used.