Figure 1

Npas4 expression during neural differentiation of ESCs. (A) RT-PCR analysis was used to determine the temporal expression profile of Npas4 mRNA in relation to various marker genes during N2B27 differentiation of mESCs (n = 3). Primers to the reference gene β-actin were used as a loading control. The negative control reaction (−) contained water in place of template cDNA. (B) The changes in Npas4 expression during N2B27 differentiation were quantified using qRT-PCR. At each time point Npas4 expression was normalized to β-actin expression and fold changes are relative to Day 0 (undifferentiated mESCs). Mean values and standard deviations of three independent experiments (n = 3) are displayed. (C) In situ hybridization analysis of Npas4 mRNA expression at Day 4 of N2B27 differentiation of mESCs. Representative images of differentiating colonies from two independent experiments (n = 2) are shown. Top panel - Npas4 antisense probe; Bottom panel - Npas4 sense probe. Scale bar = 100 μm. (D) Immunoblotting was used to determine the temporal expression profile of the Npas4 protein during N2B27 differentiation of mESCs. An antibody to the reference protein β-actin was used as a loading control (n = 3). (E) Immunoblotting was used to determine the temporal expression profile of Sox1 in the 46C cell line (using an antibody to GFP) and βIII tubulin during N2B27 differentiation of mESCs. An antibody to the reference protein β-actin was used as a loading control (n = 3). (F) RT-PCR analysis of NPAS4 mRNA in relation to various marker genes during Noggin-induced neural differentiation of hESCs (n = 3). Primers to the reference gene β-ACTIN were used as a loading control. The negative control reaction (−) contained water in place of template cDNA. hESCs, human embryonic stem cells; mESCs, mouse embryonic stem cells; NS, neurospheres.