Figure 4. Ronin inhibits differentiation independently of canonical pluripotency factors.

(A) Semiquantitative RT-PCR analysis of pluripotency factors and marker genes for all three germ layers. Ectopic expression of Ronin delayed upregulation of differentiation markers. (B) siRNA against Oct4 and Ronin was transfected into ES cells and the expression of their mRNA determined at the indicated time points by real-time quantitative PCR. Data are reported as means and standard deviations of triplicate experiments. Knockdown of Oct4 resulted in its loss, but Ronin expression was not affected. (C) Alkaline phosphatase staining of ES cells after siRNA knockdown of Oct4. EF1α-Ronin ES cells showed a substantially higher degree of self-renewal than their wildtype counterparts, demonstrating that Ronin functions independently of Oct4. (D) Quantification of result in panel C. Bars represent means and standard deviations of triplicate experiments. (E) ZBHTC4.1 [EF1α] ES cells were induced to differentiate by addition of doxycycline, while ZBHTC4.1 [EF1α-Ronin] ES cells ectopically expressing Ronin did respond to doxycyclin only minimally and did not differentiate. (F) Quantification of panel (E).