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. 2014 Jun 14;3:e02224. doi: 10.7554/eLife.02224

Figure 5. Pou3f1 increases neural lineage-specifier expression.

(A) Gene expression levels in control and in Pou3f1-knockdown ESCs differentiated in serum-free medium for 4 days. (B) Gene expression levels in control and in inducible Pou3f1-overexpressing ESCs at unbiased differentiation day 8. (C) Luciferase assays using the Sox2N2-luc enhancer in control, Pou3f1-full length, or in Pou3f1-ΔHOMEO vector-transfected HEK293 cells. (D) ChIP assay in control, Pou3f1-full length, or in Pou3f1-ΔHOMEO lentivirus-transfected P19 cells. A Pou3f1-specific antibody was used, and Pou3f1 enrichment at Sox2N2 and Sox2N1 enhancer regions was normalized to the Sox2 coding region. (E) Whole-mount in situ hybridization of cPou3f1 (a–h) and cSox2 (i–p) in early chick embryos from HH stage 3+ to HH stage 10. (F) Pou3f1 overexpression induces cSox2 expression ectopically. IRES-GFP (control vector, a and a′) or Pou3f1-IRES-GFP (b and b′) was electroporated into the epiblast layer of the chick embryos. cSox2 (blue) expression was examined by in situ hybridization (a, b, a′, b′). GFP expression (brown) indicating the electroporated field was detected by immunohistochemical assays (a′ and b′). The arrowhead marks the position-plane of the corresponding embryo transverse section below (i and ii). NC, notochord. The values represent the mean ± SD for A–D. (*p<0.05; **p<0.01).

DOI: http://dx.doi.org/10.7554/eLife.02224.013

Figure 5.

Figure 5—figure supplement 1. HOMEO domain is essential for the neural-promoting effect of Pou3f1.

Figure 5—figure supplement 1.

(A) Schematic structure of full-length and domain-deleted mutant Pou3f1 proteins. Row 1, full-length Pou3f1; Row 2, Pou3f1 without the N-terminus (ΔN, missing 1–244 amino acids); Row 3, Pou3f1 without the POU domain (ΔPOU, missing 245–324 amino acids); Row 4, Pou3f1 without the HOMEO domain (ΔHOMEO, missing 325–499 amino acids). (B) Gene expression levels in ESCs transfected with control, Pou3f1-full length or with Pou3f1-ΔHOMEO lentiviruses at differentiation day 4 in serum-free medium. The values represent the mean ± SD for B. (*p<0.05; **p<0.01).
DOI: http://dx.doi.org/10.7554/eLife.02224.014