Extended Data Fig. 3: Control experiments for KB production by cortex glia to sustain K-AM formation in MB neurons.

(a) In fed flies when Bmm expression was downregulated in cortex glia LTM was normal (F_2,27 = 0.29, P = 0.749). When RNAi expression was not induced, K-AM was normal (F_2,27 = 0.27, P = 0.768). (b) A second non-overlapping RNAi targeting Bmm (Bmm RNAi GD5139) was used to confirm the specific K-AM defect. In order to increase RNAi efficiency, Dicer2 expression was induced together with the RNAi expression in adult cortex glia using the Tub-Gal80^ts; UAS-Dcr2, R54H02 line. Inhibition of Bmm expression in adult cortex glia using Bmm RNAi GD5139 impaired K-AM in starved flies (F_2,42 = 10.70, P = 0.0002), whereas ARM or LTM in fed flies was not affected (ARM: F_2,33 = 1.55, P = 0.228; LTM: F_2,45 = 0.02, P = 0.982). Non-induced starved flies showed no K-AM defect (F_2,27 = 0.003, P = 0.997). (c) Downregulation of CPT1 expression in adult cortex glia did not affect LTM (F_2,37 = 1.32, P = 0.279). When RNAi expression was not induced, K-AM was normal (F_2,36 = 2.12, P = 0.135). (d) A second non-overlapping RNAi targeting CPT1 (CPT1 RNAi KK100935) was used to confirm the specific K-AM defect. As for Bmm RNAi GD5139, we used the Tub-Gal80^ts; UAS-Dcr2, R54H02 line to increase RNAi efficiency. Inhibition of CPT1 expression in adult cortex glia using CPT1 RNAi KK100935 impaired K-AM (F_2,45 = 8.72, P = 0.0006), whereas ARM or LTM in fed flies was not affected (ARM: F_2,45 = 0.87, P = 0.424; LTM: F_2,43 = 0.06, P = 0.939). Non-induced starved flies showed no K-AM defect (F_2,45 = 0.66, P = 0.524). (e) When HMGS expression is downregulated in the cortex, LTM was normal (F_2,43 = 1.74, P = 0.187). When RNAi expression was not induced, K-AM was normal (F_2,38 = 0.43, P = 0.656). (f) A second non-overlapping RNAi targeting HMGS (HMGS RNAi HMC04928) was used to confirm the specific K-AM defect. Inhibition of HMGS expression in adult cortex glia using this RNAi impaired K-AM (F_2,31 = 4.53, P = 0.019), whereas ARM or LTM in fed flies was not affected (ARM: F_2,32 = 1.56, P = 0.227; LTM: F_2,42 = 0.72, P = 0.494). Non-induced starved flies showed no K-AM defect (F_2,45 = 1.58, P = 0.217). n represents a group of 40–50 flies analyzed together in a behavioral assay. Data are expressed as mean ± s.e.m. with dots as individual values, and analyzed by one-way ANOVA with post hoc testing by Newman-Keuls pairwise comparisons test. Asterisks refer to the least significant P-value of post hoc comparison between the genotype of interest and the genotypic controls. **P<0.01, *P<0.05, ns: not significant.