Transgene induction with AGES varies with sex and transgene insertion site.

GFP quantification (expressed as arbitrary units, a.u.) was derived from fluorescence intensity readings of whole-body fly homogenates following a 7-day induction period on media containing different concentrations of auxin. Data are shown as female (A) and male (B) flies where AGES (Tub84B-AtTIR1-T2A-AID-GAL80-AID) was located on the second chromosome, or females (C) and males (D) where AGES was located on the third chromosome. Data are presented as violin plots (line at median) of n = 4 replicates per condition, each containing 3 flies per replicate. Note that the negative control (da-GAL4 alone) and positive control (da-GAL4>UAS-GFP alone) groups are identical in A/C and B/D, since these samples were run in parallel. Statistical comparisons were performed using one-way ANOVA with subsequent Bonferroni-corrected multiple comparison tests where *, p < 0.05 and ****, p < 0.0001.

Transgene induction with the AGES system remains permissible at older ages.

Schematic depicting the auxin feeding protocol which utilised differentially aged fly cohorts to assess GFP induction (A). GFP quantifications (expressed as arbitrary units, a.u.) were derived from fluorescence intensity readings of whole-body fly homogenates following a 7-day induction period on 10 mM auxin or control (0 mM) media. Data are shown for fly cohorts aged to 21d (B), 42d (C), and 63d (D) at the time of assay. Data are presented as violin plots (line at median) of n = 8 replicates per condition, each containing 3 flies per replicate. Statistical comparisons were performed using one-way ANOVA with subsequent Bonferroni-corrected multiple comparison tests where *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

High-dose auxin reduces body mass independent of genotype, and alters TAG and protein levels in genotype-dependent ways.

Whole body mass measurements are shown for da-GAL4>+ (A), da-GAL4,AGES>+ (B), and da-GAL4,AGES>UAS-GFP (C) female flies fed different concentrations of auxin for 7 days. Triacylglycerol (TAG) measurements are depicted for da-GAL4>+ (D), da-GAL4,AGES>+ (E), and da-GAL4,AGES>UAS-GFP (F) fly cohorts. Quantification of protein concentration are illustrated for da-GAL4>+ (G), da-GAL4,AGES>+ (H), and da-GAL4,AGES>UAS-GFP (I) groups. Data are described as per fly body weight (A-C) or normalised to per mg of fly for both TAG and protein values (D-F and G-I), where all metrics were derived from the same samples for a given genotype. Data are presented as violin plots (line at median) of n = 11-12 replicates per condition, each containing 5 flies per replicate. Statistical comparisons were performed using one-way ANOVA with subsequent Dunnett’s multiple comparison tests to corresponding 0 mM control conditions where *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

AGES shows tissue- and age-dependent expression patterns with the da-GAL4 driver.

Adult brain, abdominal fat body, and whole gut GFP channel images (green) are shown from dissected fly cohorts following a 7-day induction period on 5 mM auxin or control (0 mM) media, where flies were aged to 9d (A) and 42d (B) at the time of dissection. Dotted lines indicate traces performed on DAPI stains from the same images of the central brain, abdominal cuticle with attached adipose tissue and whole gut length. Corresponding mean GFP fluorescence intensity values (expressed relative to da-GAL4-alone-containing groups) are shown for the central brain (C), abdominal fat body (D) and whole gut (E) of 9d flies, alongside brain (F), fat body (G) and gut (H) measurements from 42d flies. Data are presented as violin plots (line at median) of n = 6-10 flies per condition. Statistical comparisons were performed using one-way ANOVA with subsequent Bonferroni-corrected multiple comparison tests where *, p < 0.05 and ****, p < 0.0001. Scale bars, 200 μm (brain), 500 μm (fat body) and 1 mm (gut).

AGES-driven expression of InRDN reduces fecundity but has unclear effects on longevity.

(A) Whole-fly mRNA levels for total InR (primers outside of K1405 sequence), InRDN (primers for K1405A) and InRWT (primers for wild-type K1405) are shown for da-GAL4,AGES>UAS-InRDN female flies following a 7-day induction period on 5 mM auxin or control (0 mM) media from day 2 to day 9 of adulthood prior to collection for qPCR. Data are presented as scatter plots (line at median) of n = 7 (0mM) or 8 (5mM) replicates per condition, each containing 2 flies per replicate. (B) Quantification of egg-laying over a 34-day period is illustrated for female flies of the indicated genotypes, treated with 5 mM auxin or control (0 mM) media. Data are depicted as the cumulative number of eggs laid per female per 24h, and presented as violin plots (line at median) of n = 10 replicates per condition, each containing 5 flies per replicate. (C-E) Lifespan curves are depicted for +>UAS-InRDN (C), da-GAL4,AGES>+ (D), and da-GAL4, AGES>UAS-InRDN (E) female flies following continual 5 mM auxin or 0 mM treatment from day 2 of adulthood. For all survival data, n > 140 deaths were counted per condition, with survival curves and p-values derived via Log-Rank test analyses. For qPCR and fecundity data, statistical comparisons were performed using unpaired t-tests and a two-way ANOVA with Bonferroni’s multiple comparison test respectively, where *, p < 0.05.

Auxin treatment reduces oxidative stress resistance and starvation resistance.

(A-C) Survival curves are depicted for +>UAS-InRDN (A), da-GAL4,AGES>+ (B), and da-GAL4,AGES>UAS-InRDN (C) female flies under H2O2 -based oxidative stress following 5 mM auxin or control treatment (0 mM) for 12 days. (D-F) Survival curves are shown for +>UAS-InRDN (D), da-GAL4,AGES>+ (E), and da-GAL4,AGES>UAS-InRDN (F) female flies under starvation following the same 5 mM auxin treatment regimen. For all survival data, n > 145 deaths were counted per condition, with survival curves and p-values derived via Log-Rank test analyses.

Full constituents of Triacylglycerol (TAG) reagent.

Quantities and corresponding product numbers are listed for each component required to make up the final TAG reagent used throughout experiments.