Fig. 9. Inhibition of cPouV and cNanog expression stops proliferation and induces cESC to differentiate. cESC were transfected with vectors allowing conditional expression of shRNA-2 and shRNA-1 against cPouV and Nanog, respectively, and of Cre-ERT2 recombinase, the activity of which was induced by 4-hydroxytamoxifen. Similar results were obtained with shRNA-3 against cNanog. The morphology of the cells targeted for cPouV (C,D) and cNanog (E,F) was strongly altered, compared with control cells (A,B) and with targeted cells for cOct6 (data not shown), in 60% of the clones (G). In these differentiated cells, the percentage of SSEA-1-positive cells was drastically reduced (H). Proliferation was also rapidly reduced as assessed by XTT proliferation on 12 independent clones followed for 4 days after tamoxifen addition (T=0 hours) (I). Gene expression analysis (J) revealed a strong decrease in expression of target genes cPouV and cNanog in contrast to a strong upregulation of Gata4, Gata6 and Cdx2 in cells expressing shRNA against cPouV, and the upregulation of Gata4 and Gata6 for those cells expressing shRNA against cNanog. In the latter case, no Cdx2 expression could be detected. A shRNA-3 against another Pou family member, Oct6, does not alter cPouV and cNanog expression by comparison with the control vector (empty). Oct6 expression cannot be detected in the cells targeted by shRNA against cNanog (J). Three independent experiments provided similar results.