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. 2009 Dec;11(4):523–533. doi: 10.1089/clo.2009.0020

FIG. 3.

FIG. 3.

Twist-2 silenced ES cells maintain an undifferentiated state with high expression of SSEA-1. (A) Real-time quantitative RT-PCR for Twist-1 and Twist-2 expression in mRNA isolated from indicated ES cells. Significant knockdown of Twist-2 mRNA is observed the stable Twist-2 shRNA, LV-siTwist-2 ES cells, whereas Twist-1 mRNA levels are unchanged compared to the control GFP shRNA, LV-siGFP ES cells. Data represent the means ± SD of triplicate well from representative of three independent experiments. (B) Flow cytometry of SSEA-1 expression on the surface of sorted YFP+ transduced ES cells or parental ES cells cultured in the presence of LIF. Experiment was repeated twice with similar results. (C) Semiquantitative RT-PCR analyses showing the comparable expression levels of pluripotent marker genes (Oct-3/4, Nanog and Sox-2) at the passage #1 of transduced ES cells grown from sorted YFP+ transduced ES cells and parental ES cells in the presence of LIF. Both LV-siTwist-2 and LV-siGFP ES cells gradually lost the expression of pluripotent marker genes in the in vitro differentiation culture without LIF at the comparable rate (passages #5 and 10). β-Actin as used as an internal control.