blocking solution (respectively 1:400 and 1:2000) for 1 h at room temperature. Sections were washed three times in PBS and were then incubated with 4’,6-diamidino-2-phenylindole (DAPI) and secondary antibodies (goat anti-rabbit AlexaFluor 594 and goat anti-mouse AlexaFluor 488, Invitrogen) diluted at 1:500 in PBS for 1 h at room temperature. After three washes in PBS, immuno- labeled sections were mounted with Gel Mount (Biomedia) and were examined with a DM 5000 microscope (Leica) equipped with a CCD CoolSnap camera (Roper Scientific). Statistical Analysis All experiments were performed at least three times, each in triplicate. Data are expressed as the means 6 S.E.M. Comparison of means was performed using test. p,0.05 is considered significant. Supporting Information Table S1 Oligonucleotide PCR primers used to generate the various human and mouse NXNL1 and NXNL2 promoter constructs. Found at: doi:10.1371/journal.pone.0013075.s001 (0.03 MB DOC) Figure S1 Relative activity of murine, human NXNL1 and NXNL2 upstream region promoter fragments in COS-1 and 661W cells. (A) Human NXNL1 constructs. (B) Mouse Nxnl1 constructs. The nucleotide positions of each construct are given in reference References 1. Hartong DT, Berson EL, Dryja TP (2006) Retinitis pigmentosa. Lancet 368: 1795–1809. 2. Kennan A, Aherne A, Humphries P (2005) Light in retinitis pigmentosa. Trends Genet 21: 103–110. 3. Goodwin P (2008) Hereditary retinal disease. Curr Opin Ophthalmol 19: 255–262. 4. Daiger SP, Bowne SJ, Sullivan LS (2007) Perspective on genes and mutations causing retinitis pigmentosa. Arch Ophthalmol 125: 151–158. 5. Sahel JA, Mohand-Sa¨ıd S, Le´veillard T (2005) Neuroprotection of photorecep- tor cells in rod-cone dystrophies: from cell therapy to cell signalling. CR Biol 328: 163–168. 6. Cronin T, Le´veillard T, Sahel JA (2007) Retinal degenerations: from cell signalling to cell therapy. Curr Gene Ther 7: 121–129. 7. Le´veillard T, Sahel JA (2010) Rod-derived cone viability factor for treating blinding diseases: from clinic to redox signaling. Sci Transl Med 2: 26ps16. 8. Le´veillard T, Mohand-Sa¨ıd S, Lorentz O, Hicks D, Fintz AC, et al. (2004) Identification and characterization of rod-derived cone viability factor. Nat Genet 36: 755–759. 9. Chalmel F, Le´veillard T, Jaillard C, Lardenois A, Berdugo N, et al. (2007) Rod- derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential. BMC Mol Biol 8: 74. 10. Reichman S, Kalathur RK, Lambard S, A¨ıt-Ali N, Yang Y, et al. (2010) The homeobox gene CHX10/VSX2 regulates RdCVF promoter activity in the inner retina. Hum Mol Genet 19: 250–261. 11. Yang Y, Mohand-Said S, Danan A, Simonutti M, Fontaine V, et al. (2009) Functional cone rescue by RdCVF protein in a dominant model of retinitis pigmentosa. Mol Ther 17: 787–795. 12. Wang XW, Liou YC, Ho B, Ding JL (2007) An evolutionarily conserved 16-kDa thioredoxin-related protein is an antioxidant which regulates the NF-kappaB signaling pathway. Free Radic Biol Med 42: 247–259. 13. Wang XW, Tan BZ, Sun M, Ho B, Ding JL (2008) Thioredoxin-like 6 protects retinal cell line from photooxidative damage by upregulating NF-kappaB activity. Free Radic Biol Med 45: 336–344. 14. Fridlich R, Delalande F, Jaillard C, Lu J, Poidevin L, et al. (2009) The thioredoxin-like protein rod-derived cone viability factor (RdCVFL) interacts with TAU and inhibits its phosphorylation in the retina. Mol Cell Proteomics 8: 1206–1218. 15. Cronin T, Raffelsberger W, Lee-Rivera I, Jaillard C, Niepon, et al. (Feb 5, 2010) Cell Death Diff 10.1038/cdd.2010.2. 16. Walther C, Gruss P (1991) Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113: 1435–1449. 17. Kumar R, Chen S, Scheurer D, Wang QL, Duh E, et al. (1996) The bZIP transcription factor Nrl stimulates rhodopsin promoter activity in primary retinal cell cultures. J Biol Chem 271: 29612–29618. 18. Chen S, Wang QL, Nie Z, Sun H, Lennon G, et al. (1997) Crx, a novel otx-like paired-homoeodomain protein, binds to and transcativates photoreceptor cell- specific genes. Neuron 19: 1017–1030. CRX Regulates RdCVF to the major putative TSS for each gene. Luciferase activity was normalized to control (pRL-TK) and the results are plotted as fold induction compared to pGL4.17 control plasmid. Values represent the average of at least three independent experiments done in triplicate. Error is shown as SEM. **p,0.01, ***p,0.001. (C) Human NXNL2 constructs. (D) Mouse Nxnl2 constructs. The nucleotide positions of each construct are given in reference to the major putative TSS for each gene. Luciferase activity was normalized to control (pRL-TK) and the results are plotted as fold induction compared to pGL4.17 control plasmid. Values represent the average of at least three independent experiments done in triplicate. Error is shown as SEM. **p,0.01, ***p,0.001. Found at: doi:10.1371/journal.pone.0013075.s002 (5.06 MB TIF) Acknowledgments We thank C. Jaillard, T. Cronin, and E. Cle´rin for help with various aspects of the project. Author Contributions Conceived and designed the experiments: SL JAS TL DJZ. Performed the experiments: SL SR CB MLN. Analyzed the data: SL SR CB TL DJZ. Contributed reagents/materials/analysis tools: OG. Wrote the paper: SL TL DJZ. 19. Furukawa T, Morrow EM, Cepko CL (1997) Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell 91: 531–541. 20. Mathers PH, Grinberg A, Mahon KA, Jamrich M (1997) The Rx homeobox gene is essential for vertebrate eye development. Nature 387: 603–607. 21. Mears AJ, Kondo M, Swain PK, Takada Y, Bush RA, et al. (2001) Nrl is required for rod photoreceptor development. Nat Genet 29: 447–452. 22. Cheng H, Khanna H, Oh EC, Hicks D, Mitton KP, et al. (2004) Photoreceptor- specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors. Hum Mol Genet 13: 1563–1575. 23. Wang QL, Chen S, Esumi N, Swain PK, Haines HS, et al. (2004) QRX, a novel homeobox gene, modulates photoreceptor gene expression. Hum Mol Genet 13: 1025–1040. 24. Hennig AK, Peng GH, Chen S (2008) Regulation of photoreceptor gene expression by Crx-associated transcription factor network. Brain Res 1192: 114–133. 25. Furukawa T, Morrow EM, Li T, Davis FC, Cepko CL (1999) Retinopathy and attenuated circadian entrainment in Crx-deficient mice. Nat Genet 23: 466– 470. 26. Livesey F, Furukawa T, Steffen MA, Church GM, Cepko CL (2000) Microarray analysis of the transcriptional network controlled by the photoreceptor homeobox gene Crx. Current Biology 10: 301–310. 27. Sohocki MM, Sullivan LS, Mintz-Hittner HA, Birch D, Heckenlively JR, et al. (1998) A range of clinical phenotypes associated with mutations in CRX, a photoreceptor transcription-factor gene. Am J Hum Genet 63: 1307–1315. 28. Wang J, Zhang H, Liang RX, Pang B, Shi QG, et al. (2007) Identification and characterization of the novel human prostate cancer-specific PC-1 gene promoter. Biophys Biochem Res Comm 357: 8–13. 29. Ikeda T, Saito T, Ushita M, Yano F, Kan A, et al. (2007) Identification and characterization of the human SOX6 promoter. Biochem Biophys Res Comm 357: 383–390. 30. Ameixa C, Brickell PM (2000) Characterization of chicken retinoid X receptor-c gene promoter and identification of sequences that direct expression in retinal cells. Biochem J 347: 485–490. 31. Graham FL, Smiley J, Russell WC, Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5". J Gen Virol 36: 59–74. 32. Tan E, Ding XQ, Saadi A, Agarwal N, Naash MI, et al. (2004) Expression of cone-photoreceptor-specific antigens in a cell line derived from retinal tumors in transgenic mice. Invest Ophthalmol Vis Sci 45: 764–768. 33. Matsuda T, Cepko CL (2004) Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA 101: 16–22. 34. Matsuda T, Cepko CL (2007) Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci USA 10: 1027–1032. 35. Zack DJ, Bennett J, Wang Y, Davenport C, Klaunberg B, et al. (1991) Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas. Neuron 6: 187–199. PLoS ONE | www.plosone.org 15 October 2010 | Volume 5 | Issue 10 | e13075