Analysis of cell cycle regulators in NS knockdown cells. (A) QPCR analysis of expression of Ccne1, Cdk2, and inhibitors of the cyclin E–Cdk2 and cyclin D–Cdk4/6 complexes after NS KD in ESCs. For the three right panels in A and the panel in E, samples for co-depletion of NS and a cell cycle inhibitor were transfected with two plasmids, one encoding both the puromycin resistance gene and an shRNA for Ns, the other encoding both the blasticidin resistance gene and an shRNA for a cell cycle inhibitor. For KD of NS alone, cells were transfected with two plasmids, one containing Ns shRNA and the puromycin resistance gene, the other expressing a scrambled shRNA and the blasticidin resistance gene. The controls were transfected with two plasmids, one encoding the puromycin resistance gene and scrambled shRNA, the other encoding the blasticidin resistance gene and the same scrambled shRNA. For the seven left panels in A and all panels in B–D, single-depletion samples were transfected with plasmids encoding the puromycin resistance gene and either a scrambled shRNA, an shRNA for Ns, or an shRNA for a cell cycle inhibitor. Selection with puromycin and blasticidin was initiated 24 h after transfection and continued for 3 d before cells were analyzed. Ns shRNA-1 was used in all experiments for KD of NS. For A–E, the 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. B, C, and D show QPCR analysis of the effect of shRNA constructs targeting p21, p27, and Rb1, respectively, in ESCs. (E) QPCR analysis of Ns mRNA in ESCs that were co-depleted of NS and the indicated cell cycle inhibitors.