Fig. 1. Establishment of NANOG overexpressing HESC clones and examination of their growth. (A, part I) Establishment of NANOG overexpressing HESC clones was verified following selection with puromycin by RT-PCR. Amplification was performed using specific primers for the NANOG transgene. GAPDH was used as a positive control. (II) The level of NANOG overexpression was analyzed by real-time RT-PCR to examine the total NANOG mRNA level in the cells. The three clones examined showed significant elevation of the mRNA level (^*P<0.05). (III) Western blot analysis verified that the elevation in the mRNA levels corresponded to an elevation at the protein level. TUBULIN was used as the loading control. (B) Cells (2x10⁴ per cm²) were seeded on plates coated with gelatin and grown either in the presence (top) or absence (bottom) of conditioned media. To analyze the growth rate of the cells, they were fixed in 0.5% glutardialdehyde after the indicated number of days. Cell staining was performed using Methylene Blue, color was extracted with 0.1 M HCl and absorbance (650 nm) was used as an indication of relative cell number. Error bars represent s.e.m.