Extended Data Figure 2: C18:0 regulates mitochondrial morphology.

a, Lipidomic (GC-FID) profiles of purified mitochondria from Elovl6 mutant 3rd instar larvae do not show major differences compared with control animals. Mitochondrial membranes from both control and mutant animals have very low levels of C18:0. Controls for purity of mitochondrial prep are shown in Extended Data Fig. 6b. b, c, Elovl6 mutant larvae do not have reduced amounts of mitochondria, quantified via levels of porin (b; representative of three biological replicates) or citrate synthase activity (c; n = 3). See Supplementary Fig. 14 for image of the uncropped full western blot. d, Elovl6 mutant larvae have fragmented mitochondria, which is rescued by dietary C18:0 supplementation. Mitochondrial morphology from fat bodies of control or Elovl6 mutant female larvae, fed control or C18:0 (10%) supplemented food, visualized with mitoGFP. Images are representative of eight areas of four larvae from each genotype and food conditions. Equivalent pictures for body wall are shown in Fig. 2a. e, Only C18:0, and not shorter, longer or desaturated fatty acids, restores mitochondrial fragmentation to control levels in HeLa cells grown in medium containing delipidated serum. Mitochondria were visualized with MitoTracker (red) (top) and mitochondrial fragmentation was quantified by normalizing the number of mitochondrial particles to total mitochondrial area (bottom) (n = 15). f, Reduced lipoic acid (LA) levels do not lead to mitochondrial fragmentation. Lipoic acid synthase (LIAS) was knocked down by RNAi in HeLa cells, leading to significantly reduced lipoic acid levels, assayed by immunoblotting of total cell lysates with antibody detecting lipoic acid (bottom left). Unlike removal of C18:0, this does not lead to mitochondrial fragmentation. Representative images (top left) are quantified (top right) (n = 6). See Supplementary Fig. 14 for image of the uncropped full western blot. g, h, HeLa cells growing in medium containing delipidated serum do not display reduced levels of protein lipoylation (g) or reduced levels of lipoylated proteins (h). HeLa cells were grown in medium containing delipidated serum for either 24 h (the same time point used for all other experiments in which mitochondrial fragmentation was assessed) (g, h), or for an extended period of time: 4 days (h). Lipoic acid levels were assayed by immunobloting total cell lysates with an anti-lipoic acid antibody (g), and levels of lipoylated proteins were assessed with specific antibodies (h). See Supplementary Fig. 15 for image of the uncropped full western blot. c, e, f, *P < 0.05, **P < 0.01, not significant (NS) P ≥ 0.05, two-tailed t-test. Error bars represent s.d. Scale bars, 10 µm.