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. Author manuscript; available in PMC: 2023 Apr 18.
Published in final edited form as: Nature. 2022 Jul 20;608(7921):209–216. doi: 10.1038/s41586-022-04960-2

Extended Data Fig. 7: Sestrin-regulated mTORC1 signaling in glial cells controls the preference of flies for leucine-containing food.

Extended Data Fig. 7:

a, Same data as in Figure 4a except that the values were not normalized to the values from the flies expressing the control shRNA from each of the indicated drivers. n=5 (da, pros attP40 shRNA; da Sesn shRNA), 8 (repo, tdc2 attP40 shRNA; vGAT Sesn shRNA), 12 (repo, esg Sesn shRNA), 15 (Elav attP40 shRNA), 16 (Elav, Mef2, ddc Sesn shRNA), 22 (Mef2 attP40 shRNA; Myo1A Sesn shRNA), 10 (ddc, Lpp attP40 shRNA; tdc2, promE Sesn shRNA), 11 (vGAT attP40 shRNA; Lpp Sesn shRNA), 9 (promE attP40 shRNA; pros Sesn shRNA), 13 (esg attP40 shRNA), 24 (Myo1A attP40 shRNA). Each point represents the ratio of the amount of two oligonucleotide barcodes per 5 flies.

b, Expression of wild-type Sestrin under repo-Gal4 driver in Sesn −/− flies is sufficient to partially rescue the leucine preference phenotype. n (repo-attP40 in wild type w1118) = 4, n (other conditions) = 8.

c, Overexpression of TSC1+TSC2 in glial cells using repo-Gal4; Tub-Gal80ts reduces the preference of flies for leucine-containing food. n (attP40) = 16, n (TSC1+2) = 19.

d, The Sesn mRNA (red) is expressed in all classified subtypes of glial cells as indicated by co-expression of a pan glial marker, Repo (green). The single cell RNA sequencing dataset is from a previous study39.

e, The knock-down of Sestrin using a pan glial cell driver (repo-Gal4) reduces the leucine preference of flies much more significantly than a knockdown using drivers for glial subtypes. The knockdown of Sestrin in cortex glial cells using the wrapper-Gal4 driver line significantly decreased the leucine preference of flies. n=8 (repo, 9.GMR50A12, 15.R85G01-Gal4 attP40 shRNA; 9.GMR50A12, 15.R85G01-Gal4 Sesn shRNA), 12 (1.GMR60F04, 2.GMR53B07, 3.GMR55B03, 4.GMR56F03, 5.GMR86E01, 6.GMR53H12, 10.Alrm-Gal4 attP40 shRNA; repo, 2.GMR53B07, 3.GMR55B03, 4.GMR56F03, 5.GMR86E01, 10.Alrm-Gal4, 14.R75H03-Gal4 Sesn shRNA), 10 (7.GMR35E04 attP40 shRNA, 1.GMR60F04 Sesn shRNA), 11 (8.GMR77A03, 11.Wrapper-Gal4, 14.R75H03-Gal4 attP40 shRNA; 6.GMR53H12, 11.Wrapper-Gal4 Sesn shRNA), 28 (12.Eaat1-Gal4 39915, 13.Mdr65-Gal4 attP40 shRNA), 9 (7.GMR35E04, 8.GMR77A03 Sesn shRNA), 24 (12.Eaat1-Gal4 39915 Sesn shRNA), 18 (13.Mdr65-Gal4 Sesn shRNA).

f, Confocal projection of wild-type female brains expressing 4MBOX-GFP fed the standard yeast-based food or starved of protein for 24 hours. Scale bar,10 μm.

Values are mean ± SD of technical replicates from a representative experiment. Data are representative of two independent experiments with similar results. Statistical analysis was performed using two-tailed unpaired t test (a, c, e), and two-way ANOVA followed by Dunnett’s multiple comparisons test (b).