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STEM_754_sm_supplFigure1.pdf643KFigure S1. Identification of MAb 4-63 antigen. (A) Extracts of biotinylated H9 hESCs were immunoprecipitated with 4-63 or no antibody (bead), followed by Western blot analysis using streptavidin-HRP. 4-63 in the non-IP section indicates that only 4-63 antibody was loaded. (B) Sequence analysis of L1CAM expressed in hESCs. The MS/MS spectrum of L1CAM obtained after trypsin digestion was analyzed by Q-TOF mass spectrometry. The precursor ion shown in the figure is m/z 673.93; resultant peaks were searched against the NCBInr database. Four tryptic peptides that matched the L1CAM isoform 2 are underlined in the sequence presented in B. The hESC-expressed L1CAM was the non-neuronal isoform because RSLE was not present in the peptide (N-DETFGEYSDNEEK-C) sequenced from the precursor ion of m/z 781.88. To confirm this result, we cloned the full-length cDNA encoding L1CAM from H9 cells and determined its entire nucleotide sequence. The L1CAM sequence lacks the YEGHH and RSLE peptides found in neural L1CAM.
STEM_754_sm_supplFigure2.tif2492KFigure S2. Specificity of L1CAM expression in undifferentiated hESCs and mESCs. (A) H9 cells and day 12-derived EBs were incubated with anti-SSEA-3, anti-SSEA-4, or L1CAM, followed by FITC-conjugated anti-rat IgM or anti-mouse IgG. Fluorescence is compared with control in each panel. (B-C) Total mRNAs were isolated from the hESCs (B) or msESC (C) cultured for 6 or 12 days, or the EBs derived at indicated days. The expression level of transcription factors essential for the maintenance of an undifferentiated state in ESCs (Nanog, Oct4, and Sox2) and three germ layer markers (ectoderm, Pax6; mesoderm, CD34; and endoderm, AFP) was assessed by RT-PCR.
STEM_754_sm_supplFigure3.tif1922KFigure S3. AP staining in H9-derived stable cell lines. (A) AP staining of H9-derived stable cell lines. (B) The graph shows the data of AP-positive cells. L1CAM depletion in hESCs significantly reduced AP staining. In contrast, L1CAM-overexpressing cells strongly expressed AP. Three independent experiments were performed in duplicate. Data are presented as means ± SD (**p < 0.01).
STEM_754_sm_supplTable1.pdf113KSupplementary Table 1.
STEM_754_sm_supplTable2.pdf76KSupplementary Table 2.
STEM_754_sm_suppldata.pdf160KSupplementary Data

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