CDK6 and CDC25A are downstream effectors of NANOG. (A) Quantitative RT-PCR analysis showing down-regulation of CDK6 and CDC25A in H1 NANOG, H9 NANOG, and hES-NCL1 NANOG sublines 42 h after the transfection of CDK6 and CDC25A siRNA. The data represent the mean ± SEM (error bars) from three experiments (one in each subline). The value for the control-transfected sample (vector only) was set to 1, and all other values were calculated with respect to this. (B) Western blotting showing down-regulation of CDK6 and CDC25A in H1 NANOG sublines. Molecular masses are indicated in kilodaltons. (C) Chart representation of the fraction of cells in S phase over time after transfection of CDK6 siRNA and synchronization by nocodazole for 18 h in the H9 NANOG subline assessed by propidium iodide staining. This figure represents an example of at least two independent experiments carried in the H9 subline. (D) Flow cytometry images showing retention of cells in G1 phase of the cell cycle after transfection of CDC25A and NANOG siRNAs and synchronization by nocodazole for 18 h in the H9 NANOG subline assessed by propidium iodide staining. This figure represents an example of at least two independent experiments performed in the H9 subline.