Fig. 3: Flies prefer to eat leucine-containing food in a fashion that depends on the capacity of Sestrin to bind leucine.

a, A schematic of the two-choice food preference assay (see Methods for details). AA, amino acids.

b, Wild-type female animals develop a preference for leucine over the course of several hours. Data show the fold-difference in relative food intake for the leucine-coated compared to water-coated apples. n≥11 per time point.

c, Rapamycin prevents flies from developing a preference for the leucine-coated apple. n≥5 per condition.

d-f, Sesn^L431E and Sesn^−/− animals fail to develop a preference for the leucine-containing apple. (d, e), n≥4 per condition; (f) n≥6 per condition.

g, Immunoblotting for Sestrin following knockdown of Sesn in adult flies. Akt serves as a loading control.

h, Ubiquitous knockdown of Sesn reduces the preference of adult female flies for leucine. Data show the fold-difference in food intake for the leucine-coated apple relative to the water-coated apple. n≥5 per condition.

i, The approach used to achieve temporal control of Sesn knockdown in (j, k).

j, Sesn immunoblot showing Gal80^ts-mediated depletion of Sestrin in adult, but not developing, animals. Extracts were prepared from flies raised at indicated temperatures. S6K serves as a loading control. Note that heat shock induces Sestrin protein levels in control flies.

k, Knockdown of Sestrin during adulthood is sufficient to decrease the preference of female flies for leucine-containing apples. n≥13 per condition.

(a, i) created with BioRender.com. (b, c, d-f, h, k), Values are mean ± SD of biological replicates from a representative experiment. Each experiment was repeated three (d-k) or two (b, c) times with similar results. Statistical analyses: one-way ANOVA followed by Dunnett’s multiple comparisons test (b), two-way ANOVA followed by Šídák’s multiple comparisons test (c-e), one-way ANOVA followed by Šídák’s multiple comparisons test (f), and two-tailed unpaired t-test (h, k).