Figure 5.

Dual inhibition of the MEK and GSK3 kinases partially rescues ESCs from the differentiation imposed by depletion of NS. ESCs were transfected with the indicated constructs as described in Fig. 2, using Ns shRNA-1 for KD of NS. Treatment with PD0325901 (PD) and CHIR99021 (CH) was initiated 24 h later. After an additional 3 d, the cells were harvested for analysis. Throughout the experiments, cells were maintained in conventional ESC media (supplemented with serum and LIF). (A) Representative morphology and AP staining of controls and NS KD cells treated with the indicated chemical inhibitors. Bars, 100 µm. (B) Quantification of undifferentiated, AP-positive ESC colonies (as defined in Fig. 2 C) with the indicated treatment. Shown are the results in two independent experiments with error bars indicating standard deviations. (C) Western analysis of cells with the indicated treatment with antibodies to ERK, phosphorylated-ERK, and β-actin. The positions of molecular mass standards (in kilodaltons) are shown.