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. 2015 Oct 8;163(2):340–353. doi: 10.1016/j.cell.2015.09.020

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© 2015 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Figure S5 — Iron Stress Selectively Depletes Linoleate from PCs, Related to Figure 6

(A) In vitro induction of neutral lipid puncta after 24 hr culture of the CNS in sugar-free saline. Note that the in vitro LipidTOX puncta are smaller than in vivo CNS lipid droplets. Scale bars: 10 μm.

(B) In vitro induction of lipid droplets is inhibited by glial-specific knockdown of PLD, Lipin, DGAT1 or Lsd-2 (repo>RNAi, control: repo > w¹¹¹⁸).

(C) In vivo induction of CNS lipid droplets by 100 mM FeCl₂ is inhibited by glial-specific knockdown of PLD, Lipin and DGAT1 (repo>RNAi, control: repo > w¹¹¹⁸).

(D) Tandem mass spectrometry of CNS phosphatidylcholines (PCs) from larvae subject to FeCl₂ stress. Three major PC mass envelopes are shown at low (left) and high (right) resolution. High-resolution images compare the relative distributions of species containing different numbers of fatty acyl carbon-carbon double bonds under control (NR) and 100 mM FeCl₂ stress. Two independent spectra for each condition are shown and the signal intensity (y axis) is normalized for each mass envelope to the species with 1 fatty acyl double bond. FeCl₂ stress strongly decreases the relative abundance of PCs with at least one C18 chain (34:x and 36:x mass envelopes) and at least two C-C double bonds (red arrows).

(E) Overlaid neutral loss scans from tandem mass spectrometry indicate that, in both control and FeCl₂ conditions, CNS TAGs contain fatty acids that are saturated (18:0, stearic acid), monounsaturated (18:1, oleic acid) and polyunsaturated (18:2, linoleic acid). Y values correspond to relative signal intensities scaled to the largest C18:1 peak.

(F) Tandem mass spectrometry shows that TAG composition does not change substantially between NR controls (n = 3), hypoxia (2.5% oxygen, n = 3) or tBH (100 mM, n = 4). Larvae were raised on the linoleate (C18:2) rich diet, and the relative abundance of the following TAG species was calculated: 16:1/16:1/16:0 (m/z 820), 16:1/16:0/16:0 (822), 16:0/16:0/16:0 (824), 18:2/16:1/16:0 (846), 18:2/16:0/16:0 (848), 18:3/18:2/16:0 and 18:2/18:2/16:1 (870), 18:2/18:2/16:0 (872), 18:1/18:2/16:0 (874), 18:1/18:1/16:0 and 18:2/18:0/16:0 (876), 18:1/18:0/16:0 (878), 18:2/18:2/18:2 (896), 18:2/18:2/18:1 (898), 18:2/18:1/18:1 and 18:2/18:2/18:0 (900). The sn1 to sn3 order of fatty acyl chains was not analyzed.