Fig. 1.

Knockdown of Geminin decreases neural fate commitment of ES cells. Gem shows significant colocalization with the neural markers Sox1 and Pax6 by day 3 of neural commitment as shown in (A) images and (C and D) quantitated as percentages of total ES cells and Gem immunopositive ES cells also immunopositive for Pax6 or Sox1 (SI Materials and Methods). (B) Dox-induced Gem knockdown (days 0–3) in a GemKD clonal line also expressing GFP. There is decreased Pax6 and Sox1 expression in GFP-expressing Gem knockdown cells (compare A and B, quantitation in E). Gem knockdown also decreases levels of (F) Pax6 and Sox1 protein and (G) Pax6, Neurod4, and Zic5 RNA as detected by immunoblotting or qRTPCR (**P < 0.01, *P < 0.05). Dox treatment of the parental A2lox ES line, which induces GFP overexpression, has no effect on neural gene expression.