Figure 2.
Analysis of Dot1L deficiency in undifferentiated ESCs harboring stably-integrated shRNA vectors. (A) RT-PCR of RNA from ESC lines shDot1L-1 and shDot1L-2 exhibited robust knockdown of Dot1L, but not Oct4 or Gapdh mRNAs. Also, Western blot analysis shows greatly diminished H3K79 methylation in ESC lines shDot1L-1 and shDot1L-2 without substantial alteration of Oct4 protein levels. The histone macroH2A was used as a loading control. (B) Quantitative real-time PCR was used to assay the expression of several genes in Dot1L-deficient ESCs. Dot1L mRNA is decreased in Dot1L knockdown ESCs compared to the empty vector control. MLL, a gene which encodes a histone methyltransferase specific for H3K4, is unaffected, as well as mRNAs expressed from Oct4, Sox2, or β-actin, which were not downregulated. Data is shown as the mean of 6 PCR reactions +/− the standard deviation. (C) Growth curve analysis of two Dot1L-deficient ESC lines as compared to a wild-type ESC control line harboring an integrated empty vector. The plotted data is the mean of 3 cell culture counts; error bars indicate standard deviation. (D) Cellular morphology of shDot1L ESCs compared to empty vector ESCs.