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Files in this Data Supplement:
Fig. S1. The expression pattern of E/nestin:dVenus and the upstream regulators Brn2 and SOX2 in the developing cerebral wall. dVenus expression was strictly localized to the ventricular zone according to the expression pattern of Brn2 and SOX2. The dVenus expression was also observed throughout other proliferative zones of the developing CNS − the outer layers of the retina, around the central canal of the spinal cord, and the developing cerebellum (data not shown). Moreover, immunohistochemical analysis showed that the fluorescence signal of dVenus was detected in neural progenitor cells expressing nestin, RC2 and Msi-1 protein (data not shown). Scale bars: E11.5 and 14.5 whole mounts, 2 mm; coronal sections, 500 µm.
Fig. S2. E/nestin-dVenus-positive fraction contained a high rate of selfrenewable and multipotent neural stem cells. (A) FACS profile of E/nestin-dVenus, E/nestin-EGFP, and wild-type mice cerebral wall derived cells. Note that most of the E/nestin-EGFP derived cells were positive compared with the wild-type control. (B) Cells from the dVenus-positive (F2) fraction formed an extremely higher rate (16±1 cells/500 cells) of neurospheres than that of F1 (0.2±0.4 cells/500 cells) and unfractionated cells (UF; 3±0.7 cells/500 cells). Sorted cells were followed by standard procedures to form neurospheres, as previously described (Reynolds and Weiss 1996). Cells were cultured in DMEM/F12 (Sigma-Aldrich) based serum-free media containing B-27 supplement (Invitrogen), EGF, FGF2 (20 ng/ml each), heparin (5 µg/ml), and gentamycin (10 µg/ml) in a clonal cell density (2.5 cells/µl). The culture media also contained a half-volume of neurosphere-conditioned medium. After 8 days culture, the number of spheres larger than 50 µm was counted. (C) Cells in the F2 fraction contained self-renewable and multipotent neural stem cells. Primary spheres formed from F2 fraction were mechanically dissociated into single cells and cultured to form secondary spheres, or attached on pre-coated coverslips in a differentiation media containing 1% fetal bovine serum (FBS) instead of mitogen for another 8 days. Differentiated neurospheres were immunostained according to a previously reported protocol (Reynolds and Weiss, 1996). Primary neurospheres generated secondary spheres and differentiated into neurons (red), astrocytes (blue), and oligodendrocytes (green). (D) The F2 fraction contained not only cells in G1, S phase, but also cells in G2-M phase. Although we showed that dVenus expression was weak in G2-M phase (Fig. 2), representative FACS profile of PI staining showed that the dVenus-positive (F2) fraction contained cells in all cell cycle phases. G1, 55.2%; S, 31.4%; G2-M, 8.13%. This result probably reflects the high sensitivity of FACS, detecting the cells that we had judged negative, visually on slide sections (Fig. 2), as positive. Scale bar: 25µm.
Fig. S3. Representative FACS profile of synchronized cells with PI staining. 293T cells were maintained in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% FBS and L-glutamin. Exponentially growing cultures of 293T cells were once synchronized at the border of G1-S phase by adding 2 mM thymidine (Sigma-Aldrich), followed by release from this blockage by placing them back into complete medium. Cells in M phase were synchronized with a following 0.1 µg/ml nocodazole (Calbiochem, San Diego, CA) treatment. To collect cells in G1 phase, an additional thymidine treatment was performed. By releasing cells from the second thymidine blockage, S phase cells were collected. The whole cell lysates of each cell cycle phase were prepared in lysis buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 30 mM sodium phosphate, 50 mM sodium fluoride, 20 mM β-glycerophosphate, 1% Triton X-100, phosphatase inhibitor cocktail 1 (Sigma-Aldrich), and protease inhibitor mixture (Complete; Roche Applied Science) for immunoprecipitation followed by electrophoresis. To determine each cell cycle phase, flow cytometric analysis was performed with samples of 106 cells. After washed twice with PBS, cells were stained with PI in a hypotonic buffer (50 µg/ml PI, 0.1% sodium citrate dihydrate, 0.2% NP-40 and 0.25 mg/ml RNase A) for 30 minutes at 4°C, followed by another 15 minutes incubation at 37°C. PI stained cells were analyzed on the Becton Dickinson FACS Calibur system using CELLQuest software.
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