TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing
Figure 3
Alkaline phosphatase analysis of stably transfected ES cells at 7 days post-LIF removal. Once at confluency stably transfected pCDNA3 and TET2-pCDNA3 E14 ES cells were plated at 1000 cells per plate on gelatin coated 10 cm tissue culture plates in triplicate. 72 hours after plating LIF was removed from the media and colonies were left to grow for a further 7 days. Analysis of alkaline phosphatase activity was conducted using the Alkaline Phosphatase Leukocyte kit (Sigma-Aldrich) and each colony was given a leukocyte alkaline phosphatase (LAPA) score. (a) Average LAPA scores of stably transfected ES cells at 7 days post-LIF removal. A total of 159 colonies were analysed for the TET2-pCDNA3 condition and 128 for the pCDNA3 condition. The LAPA score for TET2-pCDNA3 was 0.9 (inverse was 1.07), whereas pCDNA3 stable transfects had an average LAPA score of 0.45 (inverse was 2.22), showing that the TET2-pCDNA3 colonies were 50% less differentiated than the control colonies. (b) shows colonies at 7 days post-LIF removal that have been stained for alkaline phosphatase (AP) activity.
