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. 2023 Jul 10;120(29):e2303779120. doi: 10.1073/pnas.2303779120

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Copyright © 2023 the Author(s). Published by PNAS.

This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

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Fig. 5. — Adult-specific loss of PDF with AGES leads to loss of rhythmicity and wakefulness. (A) Representative images of sLNvs stained for mRFP and PDF. Upon feeding 10 mM auxin for 2 wk, there is loss of PDF staining in cells expressing both UAS-Cas9 and UAS-Pdf-g. (The scale bar represents 10 µm.) (B) Quantification of PDF staining intensity from sLNvs is represented with boxplots, n ≥ 39 cells from at least 11 hemibrains per genotype/condition. There is a significant effect of both variables on PDF staining intensity and a significant interaction when AGES and CRISPR reagents for Pdf were combined with auxin feeding (Two-way ANOVA; *** Interaction P < 0.001). (C) Actograms represent double-plotted average activity of flies from an experiment across multiple days. Yellow panels indicate lights ON. Panels in dotted boxes indicate auxin feeding. (D) Rhythmicity index for individual flies quantified (from DD2-7) represented by a boxplot, n ≥ 31 per genotype. There is a significant effect of both variables on rhythmicity and a significant interaction when AGES and CRISPR reagents for Pdf were combined with auxin feeding (Two-way ANOVA; *** Interaction P < 0.001). (E) Total sleep duration in a day for individual flies quantified (from DD1-4) represented by a boxplot. There is a significant effect of both variables on sleep duration and a significant interaction when AGES and CRISPR reagents for Pdf were combined with auxin feeding (Two-way ANOVA; *** Interaction P < 0.001). (F) p-wake over a 24-h period for individual flies quantified (from DD1-4) represented by a boxplot. There is a significant effect of both variables on p-wake and a significant interaction when AGES and CRISPR reagents for Pdf were combined with auxin feeding (Two-way ANOVA; *** Interaction P < 0.001). (G–I) Rhythmicity index for individual flies quantified (from DD2-6) represented by a boxplot, n ≥ 30 per genotype. Flies of the indicated genotypes were fed auxin for either 2, 4 or 7 d and compared with flies that were fed no auxin, or with flies with mutagenesis throughout development (NO AGES). Letters represent statistically distinct groups; Kruskal–Wallis test followed by a post hoc Dunn’s test, P < 0.003.