Figure 1

Silencing of OCT4, NANOG, and SOX2 in hEC cells. (A): Initial RNAi screen on hESC marker genes in hEC cells. esiRNA-treated samples were evaluated on the basis of cell morphology and changes in OCT4, NANOG, and SOX2 expression levels (by real-time PCR). Numbers at the bottom are array-based expression ratios of hESCs vs. universal reference RNA. The values for GDF3 and OTX2 are from an in-house platform (our unpublished data) and [34], respectively. Knock-down efficiencies were between 60 and > 90% throughout (not shown). (B): RNAi phenotypes in the OCT4, NANOG, and SOX2 knock-downs. Pictures were taken 2.5 days after esiRNA transfection. The morphology of unmanipulated or mock-treated cells was dependent on the seeding density. When plated at low density as required for esiRNA transfections the cells grew as 3D-shaped colonies rather than in monolayers. Bottom left: Growth curves of NANOG vs. GAPDH esiRNA-transfected cells. (C): Immunostaining of OCT4 protein in samples prepared as in (B). Note that the NANOG RNAi cells are OCT4 positive. (D): Western blot on day 3 RNAi and mock control samples using OCT4, NANOG, and SOX2 antibodies. GAPDH served as a loading control.