Figure 1: Pcgf6 is Essential for the Maintenance of ESC Identity.

(A) Distribution plot showing z score of Oct4-EGFP signal changes with various siRNA treatments. Oct4-EGFP ESCs were transfected with indicated siRNAs (siPcgf6, siOct4, siNanog) and EGFP signal was detected and measured by flow cytometry. Non-targeting control siRNA serves as negative control (NTC). Z score was calculated by normalizing to NTC. (B~E) Scatter plot showing mRNA expression changes in Pcgf6- or Oct4-depleted CCE ESCs one or two days after siRNA transfection. Cells were transfected with Pcgf6 or control siRNAs for 3–5 h, and mRNA expression was analyzed by microarray 1 or 2 days later. Green dots indicate genes showing reduced expression (<−0.6 on log₂ scale) in Pcgf6- or Oct4-depleted compared with control cells. Red dots indicate increased genes (>0.6 log₂) and black dots indicate non-differentially-expressed genes with Pcgf6 or Oct4 knockdown. The number of differentially expressed genes is shown in the box in each panel. (F) Table showing gene ontology analysis (GO) of decreased genes in Pcgf6-depleted ESCs. 2983 decreased genes at day 1 post Pcgf6-depletion in ESCs were analyzed using DAVID^54,55. Probability is represented as Score (−log₁₀ p-value). (G) Heat map showing mRNA expression profiling in CCE ESCs depleted of Pcgf6 or Oct4. Cells were transfected with Oct4, Pcgf6, or control siRNAs for 3–5 h, and mRNA expression was analyzed by microarray 1 day later. Results in Oct4 and Pcgf6 knockdown (KD) cells were normalized to cells treated with control siRNA and then subjected to K-mean clustering analysis. Color bar is on a log₂ scale. Red arrow indicates the small proportion of genes showing increased expression in Oct4 KD cells, but which are decreased in Pcgf6 KD cells. (H) Venn diagram showing overlap between genes suppressed by Pcgf6 or Oct4 KD. mRNA expression was analyzed by microarray 2 days after transfection of CCE ESCs with siRNA, and results were normalized to cells treated with control siRNA. The data show that 97.3% of genes that were reduced by Oct4 KD were also reduced by Pcgf6 KD.