Mouse ESC line 46C (passage 2535), which has one allele of Sox1 inactivated by targeted integration of a GFPirespac cassette, was cultivated as previously described . Briefly, ESCs were maintained on gelatin-coated dishes in the absence of feeder cells in GMEM (Sigma, St. Louis, MO) supplemented with 2 mM glutamine (Gibco, Grand Island, NY), 0.001% β-mercaptoethanol (Gibco), 1× nonessential amino acids (Gibco), 10% fetal bovine serum (FBS) (Gibco), and 2,000 units/ml human recombinant LIF (R & D Systems, Minneapolis, MN).
ESC adherent monoculture
Procedure was described in detail in . Briefly, ESCs were washed with N2B27 serum-free medium to remove serum and then plated onto 0.1% gelatin-coated tissue culture plastic at a density of 1 × 104 cells/cm2 in N2B27 with or without RA (Sigma). N2B27 consists of a 1:1 ratio of DMEM/F12 and Neurobasal media supplemented with 0.5% modified N2 (Gibco), 0.5% B27 (Gibco), and 2-mercaptoethanol (Gibco). Medium was changed every other day. For purifying Sox1GFP+ cells, 0.5 μg/ml puromycin (Sigma) was used.
For clonal cultures assay, dissociated ESCs were plated onto 0.1% gelatin-coated 6-well plates (Corning Incorporated, Corning, NY) at the density of 1.5 × 103 cells/well and cultured in serum-containing ESC medium (3 ml/well) plus LIF. At this density, more than 90% of colonies derived from single cells. After 2 days, medium was replaced with serum-free neural differentiation medium N2B27. Medium was renewed with fresh N2B27 every other day. After 2 days culture in N2B27 medium, total colonies and colonies containing Sox1GFP+ cells were counted.
Cells were counted by using a hemocytometer.
Immunofluorescence and FACS
Cells were fixed in 4% paraformaldehyde and incubated for 1 h in blocking buffer (PBS, 10% FBS, and 0.1% Triton X-100). Primary antibodies were diluted in blocking buffer and applied overnight at 4°C. After three washes in PBS, secondary antibodies conjugated to TRITC (tetramethylrhodamine isothiocyanate) (Vector, Burlingame, CA) were diluted at 1: 200 in blocking buffer and applied for 1 h at room temperature. The cells were washed at least three times in PBS and visualized on an Olympus inverted fluorescence microscope. For nuclear counter staining, nuclei were stained with bisbenzimide (Hoechst 33342, 10 μM, Sigma).
Primary antibodies were obtained from the following sources: Oct4 (1: 500, Santa Cruz Biotechnology, Santa Cruz, CA), nestin (1:200, Chemicon, Temecula, CA), Tuj1 (1:200, R & D Systems), MAP2 (1: 200, Sigma), GFAP (1:400, Chemicon).
FACS analysis was performed using a Becton-Dickinson (Palo Alto, CA) FACS Calibur flow cytometer.
RT-PCR and Q-PCR analysis
RT-PCR analysis of at least three independent cultures was performed in most of the experiments, and similar results were obtained.
RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), DNase I-digested (Invitrogen), and column-purified (RNeasy MinElute Cleanup; Qiagen, Valencia, CA). First-strand cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen). To analyze relative expression of different mRNAs, the amount of cDNA was normalized based on the signals from ubiquitously expressed GAPDH mRNA. To provide negative controls and exclude contamination by genomic DNA, the reverse transcriptase was omitted in the cDNA synthesis step, and the samples were subjected to the PCR reaction in the same manner with primer sets for GAPDH, and were indicated at the bottom of each figure as RT (-). Primer sequences and PCR cycling conditions could be found in the Additional file 7 Table S1.
Q-PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to manufacturer's instructions. Signals were detected with an ABI7300 Real-Time PCR System (Applied Biosystems). The relative expression level was determine by the 2-delta Ct method and normalized against ribosomal protein L19 (RPL19). Primer sequences could be found in the Additional file 8 Table S2.
Western blot analysis
Cells and tissues were lysed in a lysis buffer (20 mM Tris-HCl, pH 7.4, 2% Triton X-100, 10 mM EDTA, 5 mM NaF and 1 mM sodium orthovanadate). Protein concentration was determined by BCA assay (Pierce, Rockford, IL). Then proteins were resolved in 12% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were then incubated overnight at 4°C in blocking solution containing 5% nonfat dry milk in PBS with 0.1% Tween-20. Subsequently the membranes were incubated with primary antibody against Oct-4 (1:1000, Santa Cruz), ERK1/2, phosphor-ERK1/2 (both 1:500, Cell Signaling Technology, MA), Neuronal Nuclei (NeuN, 1:200, Chemicon), β-actin (1:2000, Biovision, Mountain View, CA) or alpha Tubulin (1:2000, Sigma) followed by incubation with HRP (horseradish peroxidase)-conjugated second antibodies. Detection of HRP was performed by SuperSignal West Pico Chemiluminescent Substrate (Pierce).
Ro 415253 (Biomol, Plymouth Meeting, PA) was used at 1 μM. PD184352, PD0325901 and CHIR99021 (kind gifts of Professor Qilong Ying, University of Southern California, USA) was used at a concentration of 4 μM, 4 μM and 3 μM respectively. SU5402 (Calbiochem, San Diego, CA) was used at 5 μM. LiCl (Sigma) was used at 10 mM.
The statistics were analyzed by STAT 8.0 software. For comparisons of two conditions, the Student's t test was used. All data were expressed as mean ± standard error of mean (S.E.M.).