(A) Schematic expression profiles of Pou3f1 and of several key marker genes during ESC neural differentiation in serum-free medium. Rex1, ESC marker; Fgf5, EpiSC marker; Sox1, NPC marker; Tuj1, neuron marker. Detection of Pou3f1 protein expression during ESC neural differentiation by Western blotting. (B) Gene expression levels in control-ESCs (Ctrl) and in Pou3f1-knockdown ESCs (Pou3f1-KD1, Pou3f1-KD3) at neural differentiation day 4 were determined by Q-PCR. Three independent experiments were performed. (C) Immunocytochemical assays of Sox/Oct4, Pax6, and Tuj1 in day 4 EBs described in B. DNA is stained with DAPI. Scale bars: 50 μm. (D) Statistical analysis of Sox+/Oct4−, Pax6+, and Tuj1+ cells in C. (E) Gene expression levels in control-ESCs and inducible Pou3f1-overexpressing (Pou3f1-OE) ESCs at unbiased differentiation (10%FBS) day 8 were determined by Q-PCR. Dox (2 μg/ml) was added for 8 days. (F) Immunocytochemical assays of Sox/Oct4, Pax6, Nestin, and of Tuj1 in day 8 EBs described in E. Scale bars, 50 μm. (G) Statistical analysis of Sox+/Oct4−, Pax6+, and Tuj1+ cells in F. (H) Pou3f1-knockdown ESCs were transfected with control or with Pou3f1-overexpressing lentiviruses. Gene expression levels at neural differentiation day 4 were determined by Q-PCR. The values represent the mean ± SD for B, D, E, G, and for H. (*p<0.05; **p<0.01).