(Necdin+/2) mice have a reduced performance in the rotarod test in comparison with the wildtype (Necdin+/+) mice. (EPS) Figure S2 Necdin mRNA is expressed in the spinal cord during development. (A) Immunostaining of brachial and lumbar spinal cord of Hb9::GFP E12.5 embryos using antibodies against Necdin (in red). (B) In situ hybridization analysis was conducted, as previously described (Andrieu et al., 2006), to detect Necdin mRNA in the spinal cord at indicated developmental stages. (C) Combined immunohistochemical labeling (Islet-1/-2) and in situ hybridization (Necdin) on E12.5 spinal cord sections. Scale bar in (A), (B), 100 mm and (C), 20 mm. (TIF) Figure S3 The number of Islet-1/-2 positive cells in the medio-dorsal part of the spinal cord is not modified in the Necdin-deficient embryos. (A) At E17.5 anti-Islet-1/-2 antibody recognizes ventral motoneurons and a pool of interneu- rons located in the medio-dorsal part of the spinal cord. (B) A quantification of these Islet-1/-2 positive interneurons shows no difference between both genotypes throughout the rostro-caudal level. (PDF) Figure S4 TNFR1 is expressed in the lumbar region of the spinal cord at E12.5 but not at E11.5. Immunohisto- chemistry analysis revealing the expression of TNFR1 (in red) (A and B) and Hb9 (Hb9::GFP) (in green), on different transversal sections corresponding to lumbar levels of spinal cord at E12.5 (A) and E11.5 (B). TNFR1 and Hb9 are coexpressed in ventral motoneurons at E12.5 (A) but not at E11.5 (B). Transverse sections were countered stained with DAPI (in blue). (TIFF) References 1. Sutcliffe JS, Han M, Christian SL, Ledbetter DH (1997) Neuronally-expressed necdin gene: an imprinted candidate gene in Prader- Willi syndrome [letter]. Lancet 350: 1520–1521. 2. Jay P, Rougeulle C, Massacrier A, Moncla A, Mattei MG, et al. (1997) The human necdin gene, NDN, is maternally imprinted and located in the Prader- Willi syndrome chromosomal region. Nat Genet 17: 357–361. 3. MacDonald HR, Wevrick R (1997) The necdin gene is deleted in Prader-Willi syndrome and is imprinted in human and mouse. Hum Mol Genet 6: 1873–1878. 4. Cassidy SB, Driscoll DJ (2009) Prader-Willi syndrome. Eur J Hum Genet 17: 3–13. 5. Gerard M, Hernandez L, Wevrick R, Stewart CL (1999) Disruption of the mouse necdin gene results in early post-natal lethality. Nat Genet 23: 199–202. 6. Muscatelli F, Abrous DN, Massacrier A, Boccaccio I, Le Moal M, et al. (2000) Disruption of the mouse Necdin gene results in hypothalamic and behavioral alterations reminiscent of the human Prader-Willi syndrome. Hum Mol Genet 9: 3101–3110. 7. Kuwako K, Hosokawa A, Nishimura I, Uetsuki T, Yamada M, et al. (2005) Disruption of the paternal necdin gene diminishes TrkA signaling for sensory neuron survival. J Neurosci 25: 7090–7099. 8. 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Kobayashi M, Taniura H, Yoshikawa K (2002) Ectopic expression of necdin induces differentiation of mouse neuroblastoma cells. J Biol Chem 277: 42128–42135. Necdin Protects Motoneurons from Death Figure S5 Detection of interactions between TNFR1 and the Arts1-NucB2 complex and Necdin. (A–C) Immunoflu- orescence labeling of endogenous TNFR1 (in red), Arts1, NucB2 and Necdin (in green) in primary cultures of embryonic motoneurons. Motoneurons are visualized by the expression of GFP (in blue) under the control of the Hb9 promoter. (D–H) PLA labeling pattern of TNFR1-ARTS1 pair (D), TNFR1-Nuc B2 pair (E), TNFR1-Necdin (F) pair in embryonic motoneurons (in green, Hb9::GFP). (G) As a positive control, an anti-TNFR1 primary antibody was used in combination with anti-mouse PLA PLUS and anti-mouse PLA MINUS probes. (H) As a negative control, an anti-TNFR1 primary antibody was used in combination with anti- mouse PLA PLUS and anti-rabbit PLA MINUS probes. (I) Box- and-whisker plot showing the number of PLA dots for each indicated pair (I). Scale bar, 20 mm. (TIFF) Supporting materials and methods. Text S1 (DOC) Acknowledgments The authors thank Delphine Bernard for her help in the explant cultures, Claire Sunyach, Isabelle Virard and Franc¸ois Michel for their technical advice on the in situ proximity ligation assay. We are grateful to Keith Dudley and Flavio Maina for their critical reading of the manuscript and helpful comments. Author Contributions Conceived and designed the experiments: JA CR FM. Performed the experiments: JA RS DA AR FS SG. Analyzed the data: JA RS DA SG CR FM. Wrote the paper: JA CR FM. 14. Kurita M, Kuwajima T, Nishimura I, Yoshikawa K (2006) Necdin downreg- ulates CDC2 expression to attenuate neuronal apoptosis. J Neurosci 26: 12003–12013. 15. Tcherpakov M, Bronfman FC, Conticello SG, Vaskovsky A, Levy Z, et al. (2002) The p75 neurotrophin receptor interacts with multiple MAGE proteins. 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Takazaki R, Nishimura I, Yoshikawa K (2002) Necdin is required for terminal differentiation and survival of primary dorsal root ganglion neurons. Exp Cell Res 277: 220–232. 27. Andrieu D, Meziane H, Marly F, Angelats C, Fernandez PA, et al. (2006) Sensory defects in Necdin deficient mice result from a loss of sensory neurons PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e23764