dsRNA or dPANK/fbl dsRNA (as described in Materials and Methods) to downregulate dPANK/Fbl protein levels and the cells were lysed 4 or 7 days after RNAi treatment. Immunoblots of whole cell extracts were incubated with an antibody against dPANK/Fbl, Tsr and p-Tsr. Tubulin was used as a loading control. (B) The graph illustrates the quantified levels of p-Tsr relative to untreated control cells. (TIF) Figure S2 Serum starvation or oligomycin does not lead to increased levels of phospho-Twinstar. S2 cells were left untreated, serum starved for 5 hours or treated with 100 nM oligomycin for 15 or 60 minutes. Immunoblots of whole cell extracts were probed for p-Tsr levels and the starvation effect (ATP depletion) was confirmed with an antibody against phosphorylated AMP-activated kinase. Tubulin was used as loading control. CTRL = control/untreated cells. (TIF) Figure S3 SHSY-5Y cells differentiate and show mor- phological changes upon treatment with retinoic acid. (A–C) Control SHSY-5Y cells were differentiated with RA, plated on poly-lysine and analyzed after 24 hours for their morphology (using rhodamin-phalloidin marking F-actin, red) and differenti- ation status (using b-III-Tubulin staining, green). In non treated SHSY-5Y cells no b-III-Tubulin positive cells were observed (data not shown). In RA-treated cultures undifferentiated and differen- tiated cells were visible. Undifferentiated cells showed very short filopodia, no filamentous actin was observed and b-III-Tubulin was not expressed (A and marked with white arrow heads in B). After 24 hours RA treatment most differentiated cells showed lower b-III-Tubulin expression associated with strong filamentous actin fibers (purple arrow heads) and an angular cell shape (B,C). 48 hours after seeding many differentiated cells showed an elongated shape, neurite growth and high expression of b-III- Tubulin (Figure 5C–D, main text). The presence of b-III- Tubulin positive neurites was used to quantify the effect of HOPAN on differentiation by RA and to quantify the rescue potential of pantethine (Figure 5D–G, main text). (TIF) Movie S1 CoA deficient Drosophila S2 cells fail to form actin-based lamella. dPANK/Fbl depleted S2 cells were References 1. Hall A (1998) Rho GTPases and the actin cytoskeleton. Science 279: 509–514. 2. Narumiya S, Ishizaki T, Watanabe N (1997) Rho effectors and reorganization of actin cytoskeleton. FEBS Lett 410: 68–72. 3. Leonardi R, Zhang YM, Rock CO, Jackowski S (2005) Coenzyme A: Back in action. Progress in Lipid Research 44: 125–153. 4. Zhou B, Westaway SK, Levinson B, Johnson MA, Gitschier J, et al. (2001) A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome. Nature Genetics 28: 345–349. 5. Bosveld F, Rana A, van der Wouden PE, Lemstra W, Ritsema M, et al. (2008) De novo CoA biosynthesis is required to maintain DNA integrity during development of the Drosophila nervous system. Human Molecular Genetics 17: 2058–2069. 6. Rana A, Seinen E, Siudeja K, Muntendam R, Srinivasan B, et al. (2010) Pantethine rescues a Drosophila model for pantothenate kinase-associated neurodegeneration. Proceedings of the National Academy of Sciences of the United States of America 107: 6988–6993. 7. Wu ZH, Li CH, Lv S, Zhou B (2009) Pantothenate kinase-associated neurodegeneration: insights from a Drosophila model. Human Molecular Genetics 18: 3659–3672. 8. Afshar K, Go¨nczy P, DiNardo S, Wasserman SA (2001) fumble encodes a pantothenate kinase homolog required for proper mitosis and meiosis in Drosophila melanogaster. Genetics 157: 1267–1276. 9. Bosveld F, Rana A, Lemstra W, Kampinga HH, Sibon OC (2008) Drosophila phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis. BMC Res Notes 1: 75. Coenzyme A Levels Influence Phospo-Twinstar Levels seeded in concanavalin A coated glass bottom culture dishes and imaged during cell spreading and lamella formation in phase contrast with an inverted Zeiss confocal microscope using a 636 glycerol-immersion lens. Frames were acquired every 20 seconds for 20 minutes. (AVI) Movie S2 Pantethine rescues actin-based lamella for- mation of dPANK/fbl depleted Drosophila S2 cells. Pantethine treated dPANK/Fbl depleted S2 cells were seeded in concanavalin A coated glass bottom culture dishes and imaged during cell spreading and lamella formation in phase contrast as described under Figure S4. (AVI) Movie S3 Control Drosophila S2 cells form actin-based lamella. S2 cells treated with control RNAi constructs were seeded in concanavalin A coated glass bottom culture dishes and imaged during cell spreading and lamella formation in phase contrast as described under Figure S4. 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