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Original Research: Embryonic Stem Cells/Induced Pluripotent Stem Cells
Article first published online: 14 FEB 2012
Copyright © 2011 AlphaMed Press
Volume 30, Issue 3, pages 452–460, March 2012
How to Cite
Närvä, E., Rahkonen, N., Emani, M. R., Lund, R., Pursiheimo, J.-P., Nästi, J., Autio, R., Rasool, O., Denessiouk, K., Lähdesmäki, H., Rao, A. and Lahesmaa, R. (2012), RNA-Binding Protein L1TD1 Interacts with LIN28 via RNA and is Required for Human Embryonic Stem Cell Self-Renewal and Cancer Cell Proliferation. STEM CELLS, 30: 452–460. doi: 10.1002/stem.1013
Author contributions: E.N. and N.R.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and responsibility for the integrity of the work as a whole, from inception to published article; M.R.E. and J.-P.P.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; R.Lu.: conception and design, data analysis and interpretation, and manuscript writing; J.N.: collection and assembly of data; R.A., K.D., and H.L.: data analysis and interpretation; O.R.: molecular biology and construct design; A.R.: conception and design, interpretation of results, and manuscript writing; R.L.: conception and design, interpretation of results, leader and supervision of the project, and manuscript writing. E.N., N.R., and M.R.E. contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS December 12, 2011.
- Issue published online: 14 FEB 2012
- Article first published online: 14 FEB 2012
- Accepted manuscript online: 12 DEC 2011 02:58PM EST
- Manuscript Accepted: 19 NOV 2011
- Manuscript Received: 17 JUN 2011
- ESTOOLS consortium under the Sixth Research Framework Programme of the European Union, JDRF
- Academy of Finland
- Finnish Cancer Organizations
- Turku Graduate School of Biomedical Sciences
- Ida Montin Foundation
- Finnish Cultural Foundation
Additional Supporting Information may be found in the online version of this article.
|STEM_1013_sm_SuppFig1.pdf||605K||Supplemental Figure 1: L1TD1 is highly expressed in hESCs and required for hESC selfrenewal, as well as cancer cell proliferation. A) The mean expression profile of L1TD1 in Stem Cell Matrix data (Muller FJ) in parallel with known stem cell core factors OCT-4, NANOG, and SOX-2. B) Western blot validation of L1TD1 shRNA sequences using L1TD1-V5 fusionprotein overexpression in 2102Ep EC cell line and antibody against V5. C) Validation of custom made rabbit antibody 3048 in 293T cells. L1TD1-V5 fusionprotein was overexpressed in 293T cells where overexpression produces a specific band which is responding to shRNA treatment. D) Real-time PCR analysis of L1TD1 and known stem cell markers in retinoic acid induced early differentiation serie (hESC line H9). Data from two biological replicates. E) Ingenuity pathway analysis of genome-wide gene expression data indicating the cellular processes that were influenced most in response to stable L1TD1 silencing after 6 days in EC cell line NT2D1. F) Non-Radioactive Cell Proliferation assay performed with siL1TD1 and non-targeting siRNA control 1-8 days after silencing in seminoma cell line (TCam2).|
|STEM_1013_sm_SuppFig2.pdf||128K||Supplemental Figure 2: Flow cytometry analysis of pluripotency (SSEA-3, TRA-1-60) and differentiation (A2B5 and SSEA-1) markers one and three days after knockdown of OCT4 in relation to the non-targeting control (hESC line H9). Average data from two replicate cultures.|
|STEM_1013_sm_SuppFig3.pdf||288K||Supplemental Figure 3. Alignment of the putative RNA-binding region of human L1TD1 with human pORF1. RNA-recognition-motif (RRM), C-terminal domain (CTD). Conserved residues boxed in red. Residues forming the conserved salt bridges are shaded in blue. Residues providing aromatic, RNA- binding side-chains in canonical RRMs are shaded in yellow. Asterisk (*) mark residues mutated that have strong or moderate effect on RNA-binding. Data for pORF1 .|
|STEM_1013_sm_SuppFig4.pdf||383K||Supplemental Figure 4. Validation of L1TD1 location. A) Endogenous L1TD1 (green) stained in hESCs with polyclonal antiserum raised against total protein antigen. B) Preserum (taken before immunization) staining (negative control). C) Anti-V5 staining of overexpressed V5-tagged L1TD1 (red) in teratocarcinoma cells (2102Ep). D) EGFP-tagged overexpression of L1TD1 in hESCs (line HS401). E) L1TD1 (red) in hESCs co-stained with pluripotency surface marker TRA-1-60 (green) co-cultured with human foreskin fibroblast. DAPI staining (blue) used to visualize nucleus in all stainings. F) L1TD1 (red) double staining with P-body marker GW182 (green) in TCam2 cells G) L1TD1 (green) double staining with P-body marker DCP1A (red) in TCam2 cells H) L1TD1 (green) double staining with SG marker TIA1 (red) in TCam2 cells I) L1TD1 (red) double staining with endosome marker EEA1 (green) in TCam2 cells. Co-Locations shown in white.|
|STEM_1013_sm_SuppTab1.pdf||60K||Supplementary Table 1|
|STEM_1013_sm_SuppTab2.pdf||51K||Supplementary Table 2|
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