Extended Data Fig. 5: Chronic activation of JAK–STAT signalling in EG drives the decline of EG numbers at the AL during ageing.

a, Representative images of glomerular compartments at the AL from young and old flies. Confocal images were generated from 20-μm z-sections (1 μm each) after performing maximal intensity projection. Neuropils labelled by anti-NC82 antibody. b, Representative single z-section images showing EG morphology at the AL from young and old flies. EG nuclei were labelled by RedStinger driven by GMR56F03::Gal4, and cellular processes were labelled by CD4::GFP. Average intensity levels of CD4::GFP and AL sizes were quantified from 20-μm z-stack confocal images after maximal intensity projection. AL sizes were quantified and normalized to the mean values of young flies. c, Representative images of 2xSTAT::GFP reporter activity in EG (nls.mCherry⁺ driven by GMR56F03::Gal4) at the AL from young and old flies. Images were generated from 20-μm z-sections (1 μm each) after performing maximal intensity projection. d, Preference index of young flies expressing mCherry^RNAi in EG (GMR56F03::Gal4), old flies expressing mCherry^RNAi, dome^RNAi or stat^RNAi in EG or all glia (repo::Gal4), measured by T-maze assay. e, Representative images showing EG morphology at the AL from old flies with or without dome knockdown. EG nuclei were labelled by RedStinger in the presence of GMR56F03::Gal4;tubG80^ts, while cellular processes were labelled by CD4::GFP. Average intensity levels of CD4::GFP and numbers of RedStinger⁺ cells per AL were quantified. AL sizes were quantified and normalized to the mean value of old control flies. Flies were aged at 25 °C for 14 days followed by 29 °C for 14 days to induce dome^RNAi expression. f, Representative images showing the activity of 2xSTAT::GFP reporter in the central brain of old flies knocking down upd2 or upd3 in enterocytes, driven by Mex1::Gal4;tubG80^ts. Representative images were generated from 30-μm z-sections, and the AL region was magnified. g, Preference index of young flies expressing mCherry^RNAi and old flies expressing mCherry^RNAi, Upd2^RNAi or Upd3^RNAi in enterocytes, driven by Mex1::Gal4;tubG80^ts. Data are mean and s.e.m. The sample size is as follows: n = 8 and 6 brains for young and old conditions, respectively, in b, n = 6, 7, 8 and 7 independently performed experiments for young mCherry^RNAi, old mCherry^RNAi, dome^RNAi and stat^RNAi correspondingly in d (left), n = 4, 3 and 3 independently performed experiments for old mCherry^RNAi, dome^RNAi and stat^RNAi correspondingly in d (middle and right), n = 7 and 10 brains for mCherry^RNAi, dome^RNAi, respectively, in e, n = 5, 7, 7 and 7 independently performed experiments for young mCherry^RNAi, old mCherry^RNAi, Upd2^RNAi and Upd3^RNAi, respectively, in g. Data in a–c, f are representative of two independently performed experiments; data in e are representative of three independently performed experiments. P values from two-tailed Mann–Whitney test in b, d (when comparing young and old mCherry^RNAi), e, g (when comparing young and old mCherry^RNAi); P values from Dunn’s multiple comparisons test in d (right); other P values from Kruskal–Wallis test.

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