S 0, 6 and 10. For the culture of EOCs, mononuclear cells were resuspended in 12 mL M199 medium with the same supplements and were cultured under the conditions as above. Non-adherent cells were discarded 24 h later, and adherent cells were rinsed once with complete M199 medium, and fresh complete M199 medium was added to each well. First medium change 25033180 was performed 3 daysFACS analysisSingle cell suspensions were prepared from cultured or freshly isolated cells. Cells (36105) were stained with antibodies for 30 min on ice, and were analyzed by using a 
FACSCaliburTM (BD Immunocytometry Systems, San Jose, CA). Data were analyzed by using the CellQuestTM software. The antibodies and reagents used in FACS analyses included anti-mouse-CD133-FITC, anti-mouseCD34-PE, biotinylated anti-mouse-Flk-1, streptavidin-APC and anti-CD31 (BD PharMingen, San Diego, CA). Dead cells were excluded by propidium iodide (PI) staining. For cell proliferation assay, cells (16106 for EEPCs, 36105 for EOCs) were seeded in 6-well plates and were labeled with CFSE (Sigma) for 30 min. Cells were cultured for two more days, and cell proliferation was examined by FACS.Quantitative RT-PCRTotal RNA was extracted from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Complementary DNA was prepared by using a reverse transcription kit from TOYOBO (Osaka, Japan). Realtime PCR was performed by using a kit (SYBR Premix EX Taq, Takara) and the ABI PRISM 7300 real time PCR system, with bactin as an internal control. Primers used in real time PCR were as follows: b-actin, CATCCGTAAAGACCTCTATGCCAAC andNotch Regulates EEPCs and EOCs DifferentiallyATGGAGCCACCGATCCACA; CXCR4, GAAGTGGGGTCTGGAGACTAT and TTGCCGACTATGCCAGTCAAG.StatisticsThe statistical analysis was performed with the SPSS 11.0 program. Results were expressed as the means 6 SD. Comparison between groups was undertaken using the unpaired Student’s t test. P,0.05 was considered statistically significant.Migration assayChemotaxis experiments were performed in polycarbonate transwell inserts (8 mm pore, Corning Costar Corp.). SDF-1a (Peprotech) was added in the lower chamber at the concentration of 100 ng/ml. Cells were seeded at a density of 1.56105 per well in the upper compartment and were cultured at 37uC for 14 h. Non-migrating Cells were removed from the upper surface by gentle scrubbing. Migrating cells attached to the lower membrane stained with 0.1 crystal violet and were counted in five random fields.buy Pleuromutilin Supporting InformationFigure S1 Culture of EEPCs and EOCs. EEPCs and EOCs were cultured as described in Methyl linolenate web Materials and methods, and cells were photographed under a phase-contrast microscope. Magnifications, 6200. (TIF) Figure S2 Sprouting and tube formation of EOCs attached to Cytodex 3 microcarrier beads. For methods, see the Materials and methods section of the text. Beads are 70 to 150 mm in 
diameter. (TIF) Figure S3 RBP-J deficiency attenuated 16574785 cell proliferation and increased apoptosis after the transfusion of EEPCs during liver regeneration after PHx. Mice were subjected to PHx and were transfused with EEPCs derived from the RBP-J+/2 or the RBPJ2/2 mice. Cell proliferation and apoptosis in the livers of the recipient mice was determined on day 3, 5 and 7 after the transfusion by using anti-Ki67 and TUNEL staining, respectively. Ki67+ round nuclei and TUNEL+ cells were counted under microscope. Comparison of the number of cells was shown in Figure 6A. (TIF) Figu.S 0, 6 and 10. For the culture of EOCs, mononuclear cells were resuspended in 12 mL M199 medium with the same supplements and were cultured under the conditions as above. Non-adherent cells were discarded 24 h later, and adherent cells were rinsed once with complete M199 medium, and fresh complete M199 medium was added to each well. First medium change 25033180 was performed 3 daysFACS analysisSingle cell suspensions were prepared from cultured or freshly isolated cells. Cells (36105) were stained with antibodies for 30 min on ice, and were analyzed by using a FACSCaliburTM (BD Immunocytometry Systems, San Jose, CA). Data were analyzed by using the CellQuestTM software. The antibodies and reagents used in FACS analyses included anti-mouse-CD133-FITC, anti-mouseCD34-PE, biotinylated anti-mouse-Flk-1, streptavidin-APC and anti-CD31 (BD PharMingen, San Diego, CA). Dead cells were excluded by propidium iodide (PI) staining. For cell proliferation assay, cells (16106 for EEPCs, 36105 for EOCs) were seeded in 6-well plates and were labeled with CFSE (Sigma) for 30 min. Cells were cultured for two more days, and cell proliferation was examined by FACS.Quantitative RT-PCRTotal RNA was extracted from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Complementary DNA was prepared by using a reverse transcription kit from TOYOBO (Osaka, Japan). Realtime PCR was performed by using a kit (SYBR Premix EX Taq, Takara) and the ABI PRISM 7300 real time PCR system, with bactin as an internal control. Primers used in real time PCR were as follows: b-actin, CATCCGTAAAGACCTCTATGCCAAC andNotch Regulates EEPCs and EOCs DifferentiallyATGGAGCCACCGATCCACA; CXCR4, GAAGTGGGGTCTGGAGACTAT and TTGCCGACTATGCCAGTCAAG.StatisticsThe statistical analysis was performed with the SPSS 11.0 program. Results were expressed as the means 6 SD. Comparison between groups was undertaken using the unpaired Student’s t test. P,0.05 was considered statistically significant.Migration assayChemotaxis experiments were performed in polycarbonate transwell inserts (8 mm pore, Corning Costar Corp.). SDF-1a (Peprotech) was added in the lower chamber at the concentration of 100 ng/ml. Cells were seeded at a density of 1.56105 per well in the upper compartment and were cultured at 37uC for 14 h. Non-migrating Cells were removed from the upper surface by gentle scrubbing. Migrating cells attached to the lower membrane stained with 0.1 crystal violet and were counted in five random fields.Supporting InformationFigure S1 Culture of EEPCs and EOCs. EEPCs and EOCs were cultured as described in Materials and methods, and cells were photographed under a phase-contrast microscope. Magnifications, 6200. (TIF) Figure S2 Sprouting and tube formation of EOCs attached to Cytodex 3 microcarrier beads. For methods, see the Materials and methods section of the text. Beads are 70 to 150 mm in diameter. (TIF) Figure S3 RBP-J deficiency attenuated 16574785 cell proliferation and increased apoptosis after the transfusion of EEPCs during liver regeneration after PHx. Mice were subjected to PHx and were transfused with EEPCs derived from the RBP-J+/2 or the RBPJ2/2 mice. Cell proliferation and apoptosis in the livers of the recipient mice was determined on day 3, 5 and 7 after the transfusion by using anti-Ki67 and TUNEL staining, respectively. Ki67+ round nuclei and TUNEL+ cells were counted under microscope. Comparison of the number of cells was shown in Figure 6A. (TIF) Figu.