Isolation of <i>Mst-/-</i> ES cells.

<p>(A) Genotyping of wild type (WT) ES cells and <i>Mst-/</i>- ES cells derived from blastocysts by PCR amplification of genomic DNA. Wild type ES cells showed a larger band while <i>Mst-/</i>- ES cells displayed a smaller band. <i>Actin</i> was used as an internal control. (B) Phase contrast microscopy of wild type (WT) and two independent <i>Mst-/</i>- knockout ES cell lines (<i>Mst-/-</i>1 and <i>Mst-/-</i>2) grown on 0.2% gelatin in 2i+LIF medium (Upper). These cells were stained for alkaline phosphatase (Lower). Scale bar, 200 μm. (C) mRNA level of <i>Mst1</i> and <i>Mst2</i> in wild type ES cells and <i>Mst-/</i>- ES cells examined by quantitative real-time PCR using primers flanking the deleted region of <i>Mst</i>1 and <i>Mst</i>2. The data are shown as the mean ± S.D (n=3). <i>Actin</i> was normalized as an internal control. Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting analysis of the expression of Mst1 and Mst2 in wild type ES cells and <i>Mst-/</i>- ES cells. Gapdh1 was used as a loading control.