Depleting inhibitors of the cyclin E–Cdk2 complex restores ESC properties in NS knockdown cells. ESCs were transfected with the indicated constructs as described for co-depletion samples in Fig. 3 A, using Ns shRNA-1 for KD of NS. (A) Representative morphology and AP staining of ESCs after the indicated transfections. Bars, 100 µm. (B) Quantification of undifferentiated, AP-positive ESC colonies (as defined in Fig. 2 C) after the indicated transfections. Shown are the results in two independent experiments with error bars indicating standard deviations. (C) Cell cycle profiles of the cells represented in A. (D) QPCR analysis of differentiation markers Ednra (ectoderm), T and Msx1 (mesoderm), and Krt18 and Eomes (TE) in ESCs after the indicated transfections. For D and E, the RNA data were normalized to the expression of Gapdh and are represented as mean ± SEM with n = 3. The data are displayed relative to results with scrambled shRNA-transfected controls. (E) QPCR analysis of markers specific for endoderm (Gata4), mesoderm (Nkx2.5), TE (Cdx2), and ESCs (Tcl1, Eras, and Zfp42) in ESCs after the indicated transfections.