(University of Utrecht) following the protocol of Tokayasu [32,33]. Sections were imaged on an FEI 120 kV Spirit Biotwin equipped with a F415 CCD camera. Supporting Information Figure S1 MALDI-TOF MS profiling of AB2.2, NN5 and F10 N-glycans. N-glycans were permethylated prior being subjected to MS analysis and m/z values correspond to [M+Na]+ ions. For clarity reason, not all the peaks are labelled on these spectra but the m/z value and the annotations of all detected peaks are shown in Supplementary Figure 2. Grey boxes highlight the two regions detailed in Figures 7 and 8. (TIF) Figure S2 Assignments of the molecular ions observed in the N-glycan profiles of AB2.2, NN5 and F10 cells. Scheme assignments are based on the precise fit between composition calculations and the m/z (z = 1) ratio of the molecular ions detected. Ions with m/z in bold red are specific to F10 cells only. Schemes represent the most likely structures taking into account the biosynthetic pathways and the enzyme repertoire of murine cells and selected MS/MS analyses. (TIF) Table S1 Splinkerette, sequencing and PCR primers. Details of all splinkerette, sequencing and PCR primers utilised to generate vectors and confirm gene trap mutations. (DOC) References 1. Wu JH, Herp A, Wu AM (1993) Defining carbohydrate specificity of Ricinus communis agglutinin as Gal beta 1R4GlcNAc (II).Gal beta 1R3GlcNAc (I).Gal alpha 1R3Gal (B).Gal beta 1R3GalNAc (T). Mol Immunol 30(4): 333–9. 2. Wu JH, Singh T, Herp A, Wu AM (2006) Carbohydrate recognition factors of the lectin domains present in the Ricinus communis toxic protein (ricin). Biochimie 88(2): 201–17. 3. Bonifacino JS, Rojas R (2006) Retrograde transport from endosomes to the trans-Golgi network. Nat Rev Mol Cell Biol 7(8): 568–79. 4. Hughes RC (1979) How do toxins penetrate cells? Nature 281(5732): 526–7. 5. Roberts LM, Lord JM (2004) Ribosome-inactivating proteins: entry into mammalian cells and intracellular routing. Mini Rev Med Chem 4(5): 505–12. 6. Sandvig K, van Deurs B (1999) Endocytosis and intracellular transport of ricin: recent discoveries. FEBS Lett 452(1–2): 67–70. 7. Wesche J, Rapak A, Olsnes S (1999) Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol. J Biol Chem 274(48): 34443–9. 8. Guo G, Wang W, Bradley A (2004) Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells. Nature 429(6994): 891–5. 9. Wang W, Bradley A (2007) A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells. Genome Biol 8(4): R48. 10. Wang W, Bradley A, Huang Y (2009) A piggyBac transposon-based genome- wide library of insertionally mutated Blm-deficient murine ES cells. Genome Res 19(4): 667–73. 11. Thibault ST, Singer MA, Miyazaki WY, Milash B, Dompe NA, et al. (2004) A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat Genet 36(3): 283–7. 12. Wang W, Lin C, Lu D, Ning Z, Cox T, et al. (2008) Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc Natl Acad Sci U SA 105(27): 9290–5. 13. Cary LC, Goebel M, Corsaro BG, Wang HG, Rosen E, et al. (1989) Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172(1): 156–69. 14. Soriano P, Friedrich G, Lawinger P (1991) Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells. J Virol 65(5): 2314–9. 15. Powell LD (2001) Inhibition of N-linked glycosylation. Curr Protoc Immunol Chapter 8: Unit 8 14. 16. Wada Y, Dell A, Haslam SM, Tissot B, Canis K, et al. (2007) Comparison of the methods for profiling glycoprotein glycans–HUPO Human Disease Glycomics/ Proteome Initiative multi-institutional study. Glycobiology 17(4): 411–22. Man2, a1 and Ricin Resistance in ES Cells Table S2 Assignments of molecular ions [M+Na]+ observed in the MALDI-TOF MS spectra of reduced and permethylated O-glycan derived from AB2-2, NN5 and F10 cells. NeuAc: N-Acetylneuraminic acid, Hex: Hexose, HexNAc: N-Acetylhexosamine. (DOC) Table S3 Assignments of molecular ions [M+Na]+ observed in the MALDI-TOF MS spectra of deuteror- educed and permethylated glycosphingolipids derived from AB2-2, NN5 and F10 cells. (DOC) Acknowledgments The authors wish to acknowledge fellow team members in: The Wellcome Trust Sanger Institute, the Division of Molecular Biosciences at Imperial College, The Biomedical Sciences department at DSTL, the School of Biosciences at Exeter University and Strathclyde Institute of Pharmacy and Biomedical sciences department for their support and contribution to discussions. AD and SMH also acknowledge the Biotechnology and Biological Science Research Council Core Support (BBF0083091). Author Contributions Conceived and designed the experiments: WW CBH SH JY AB GD. Performed the experiments: WW CBH DG JY SH BT RR. Analyzed the data: WW CBH DG SH BT ALT. Contributed reagents/materials/ analysis tools: CL SM RT SH BT AD. Wrote the paper: WW CBH AB GD. 17. Fong WP, Wong RN, Go TT, Yeung HW (1991) Minireview: enzymatic properties of ribosome-inactivating proteins (RIPs) and related toxins. Life Sci 49(25): 1859–69. 18. Yusa K, Horie K, Kondoh G, Kouno M, Maeda Y, et al. (2004) Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom’s syndrome gene. Nature 429(6994): 896–9. 19. Meager A, Ungkitchanukit A, Nairn R, Hughes RC (1975) Ricin resistance in baby hamster kidney cells. Nature 257(5522): 137–9. 20. Hughes RC, Mills G (1983) Analysis by lectin affinity chromatography of N- linked glycans of BHK cells and ricin-resistant mutants. Biochem J 211(3): 575–87. 21. Hughes RC, Mills G (1985) Hybrid sialylated N-glycans are minor constituents of normal BHK-cell glycoproteins and a prominent feature in glycoproteins of some ricin-resistant cell lines. Biochem J 226(2): 487–98. 22. Hughes RC, Mills G (1986) Functional differences in the interactions of glycosylation-deficient cell lines with fibronectin, laminin, and type IV collagen. J Cell Physiol 128(3): 402–12. 23. Velasco A, Hendricks L, Moreman KW, Tulsiani DR, Touster O, et al. (1993) Cell type-dependent variations in the subcellular distribution of alpha- mannosidase I and II. J Cell Biol 122(1): 39–51. 24. Akama TO, Nakagawa H, Wong NK, Sutton-Smith M, Dell A, et al. (2006) Essential and mutually compensatory roles of {alpha}-mannosidase II and {alpha}-mannosidase IIx in N-glycan processing in vivo in mice. Proc Natl Acad Sci U S A 103(24): 8983–8. 25. Hato M, Nakagawa H, Kurogochi M, Akama TO, Marth JD, et al. (2006) Unusual N-glycan structures in alpha-mannosidase II/IIx double embryos identified by a systematic glycomics approach based on two-dimensional LC mapping and matrix-dependent selective fragmentation method in MALDI- TOF/TOF mass spectrometry. Mol Cell Proteomics 5(11): 2146–57. 26. Crispin M, Chang VT, Harvey DJ, Dwek RA, Evans EJ, et al. (2009) A human embryonic kidney 293T cell line mutated at the Golgi {alpha}-mannosidase II locus. J Biol Chem 284(32): 21864–95. 27. Ramirez-Solis R, Davis AC, Bradley A (1993) Gene targeting in embryonic stem cells. Methods Enzymol 225: 855–78. 28. Mikkers H, Allen J, Knipscheer P, Romeijn L, Hart A, et al. (2002) High- throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat Genet 32(1): 153–9. 29. Parry S, Ledger V, Tissot B, Haslam SM, Scott J, et al. (2007) Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Le(x) in mice. Glycobiology 17(6): 646–54. 30. Sutton-Smith M, Dell A (2006) Analysis of carbohydrates/glycoproteins by mass spectrometry, in Cell Biology: a laboratory handbook JE. Celis, ed. Academic Press: San Diego, CA. pp 425–435. PLoS ONE | www.plosone.org 12 August 2011 | Volume 6 | Issue 8 | e22993