Zheng et al. 10.1073/pnas.0701599104. |
Fig. 7. Overexpression of foxo produces arrhythmic behavior. An EP element insertion in the promoter region of foxo did not affect rhythms (12 of 15 flies were rhythmic), but when it drove expression of foxo under the control of a tim-Gal4 (TG) driver, flies became arrhythmic (all 22 flies tested displayed arrhythmic behavior). In situ hybridization and RNase protection assay confirmed that foxo was overexpressed under these conditions and that expression of clock proteins was reduced in lateral neurons (data not shown). Because foxo mutants do not have a circadian behavioral phenotype under normal conditions, we suspect that the arrhythmicity is caused by repressor activity of ectopically overexpressed FOXO.
Fig. 8. Paraquat (PQ) treatment does not affect the phototaxis behavior of foxo mutants. Three- to 5-day-old male flies were treated with 1 mM PQ for 3 days and subsequently tested for phototaxis behavior in a dark room using a simplified countercurrent apparatus. Flies that moved to the lit end of the tube within 1 min were isolated from cycle 1 and tested again in cycle 2 [Benzer S (1967) Proc Natl Acad Sci USA 58:1112-1119]. The percentages of flies that moved toward light, averaged from two experiments, are shown. Total number of flies: WT, no PQ = 159; with PQ = 141; foxo-/-, no PQ = 141; with PQ = 94).
Fig. 9. PQ treatment dramatically alters excitation properties of roGFP. Drosophila S2 cells were transfected with 500 ng of pAct-roGFP (pCMV-roGFP was kindly provided by Toshinori Hoshi, and roGFP was subcloned into the pAC5.1 vector) in a six-well plate using cellfectin (Invitrogen). Twelve hours after transfection, cells were treated with 300 mM PQ for 48-72 h. Confocal imaging of roGFP was performed by using an Olympus FluoView (FV1000, ver.1.3c) with the following settings: excitation wavelength 405 nm and 488 nm, a 405/488 dichroic mirror, and an emission filter 521BF29. Under normal conditions, roGFP emits a strong signal when excited with a wavelength of 488 nm and a low signal when excited at 405 nm; in an oxidized environment the emission from 488 nm excitation is reduced and from 405 nm is increased. This shift was observed in the 48-h PQ-treated cells (similar results were obtained from 72-h PQ treatment), indicating that the cellular environment was shifted toward a more oxidized state [Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY (2004) J Biol Chem 279:22284-22293].
Fig. 10. FOXO is not detectable in the central clock cells. (A) FOXO is not detected in the small ventral lateral neurons even after treatment with PQ. Flies were treated with 1 mM PQ for 24 h, and brains were dissected at ZT 1. PER, green; FOXO, red. (B) foxo-Gal4 drives UAS-GFP expression in the fat body but not in the central clock cells.
Fig. 11. Expression pattern of FOXO in the rescue lines. FOXO expression driven by DJ634 is not detectable in lateral neurons. In contrast, FOXO is expressed in the lateral neurons when it is driven by Pdf-Gal4. (Left) Whole brain. (Right) LNvs.
Fig. 12. Induction of INR expression in the adult fat body tissue increases sensitivity to PQ. Behavioral rhythms were monitored in locomotor tubes containing 0.5 mM RU486 and 1 mM PQ. Induction of INR expression in the adult fat body, using the S106 driver, increased sensitivity of behavioral rhythms to PQ. Treatment of 0.5 mM RU486 or 1 mM PQ alone had no effect (data not shown).
Fig. 13. als mRNA levels are increased in foxo mutants. Absolute readings from quantitative PCR experiments were normalized to levels of actin. Three different allelic combinations of foxo mutants and their respective controls are shown. Pairwise comparisons are as follows: CS, foxo-/Df; y w, foxo21/25; CTL, foxo25.