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. Author manuscript; available in PMC: 2011 Feb 5.
Published in final edited form as: Cell Stem Cell. 2010 Feb 5;6(2):153–166. doi: 10.1016/j.stem.2009.12.014

Figure 2. Induced expression of PCL2 partially rescues knockdown phenotype.

Figure 2

A) Two Pcl2 shRNA clones containing the doxycycline inducible Pcl2-IRES-βgeo expression cassette were stained for β-galactosidase expression before and 72 hours after addition of doxycycline to the media. B) By Western analysis, PCL2 protein was increased in the two rescue clones following doxycycline induction. C) By qPCR analysis, Pcl2 transcript was increased following doxycycline induction. D&E) The rescue clones were plated in +LIF or -LIF, with or without doxycycline and the distribution of colonies on each plate that were undifferentiated or fully differentiated were determined by morphology and alkaline phosphatase (ALP) expression. F) The percentage of cells in the rescue clones that had lost OCT4 expression by 72 hours in both +LIF and −LIF, with or without doxycycline. G) Relative transcript levels of ESC specific, self-renewal genes before and after long term culture of rescue clones in doxycycline. Expression levels are reported as a percentage of expression in wild-type R1 cells.