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. 2016 Sep 20;7:12871. doi: 10.1038/ncomms12871

Figure 6. Inhibition of TOR in adult glia inhibits Draper and glial clearance of axon debris in young animals.

Figure 6

(a) Single confocal slices of antennal lobe regions. Brains immunostained with Draper. (b) Quantification of cortical Draper immunostainings shown in a; mean±s.e.m. plotted; ****P<0.0001, one-way analysis of variance (ANOVA) with Sidak post-hoc test; N≥16. (c) Quantitative PCR (qPCR) analysis of draper-I transcript levels in adult heads; mean±s.e.m. plotted, NS, not significant, one-way ANOVA with Sidak post-hoc test; N=3 biological replicates. (d) Representative Z-stack projections of OR85e GFP+ axons. (e) Quantification of GFP+ axonal debris shown in d; mean±s.e.m. plotted; *P<0.05 and ****P<0.0001, one-way ANOVA with Sidak post-hoc test. N=22. (f) Polysome profiles for young and aged whole head lysates resolved on a 10–60% sucrose gradient. Absorbance continuously monitored at 254 nm during fractionation shown. (g) draper-I mRNA in each fraction was quantified by qPCR and normalized to the housekeeping gene Rpl32 and luciferase (control for RNA recovery). mean±s.e.m. plotted; *P<0.05 and ****P<0.0001, two-way ANOVA with uncorrected Fisher's LSD post-hoc test. N=3 groups of 30 w118 fly heads/age. (h) Normalized Rpl32 Ct values pooled from four experiments on young and aged w118 brain lysates; mean±s.e.m. plotted, NS, not significant, unpaired t-test. N=17 biological replicates/age. (i) Representative western blotting for Rpl32 (green) and MemCode total protein stain (bottom panel) performed on head lysates from young and aged flies. (j) Quantification of Rpl32 western blottings; unpaired t-test. N=4 biological replicates/age. Genotypes: a–e, Control=w1118;OR85e-mCD8::GFP, tubulin-Gal80ts/+; repo-Gal4/+. D/N TOR=w1118;OR85e-mCD8::GFP, tubulin-Gal80ts/+; repo-Gal4/UAS-D/N TOR. TOR RNAi=w1118;OR85e-mCD8::GFP, tubulin-Gal80ts/+; repo-Gal4/UAS-TORRNAi. f–j, w1118. Scale bars=30 um.