Figure 6.

Functional analysis of NR2F2 in hESCs. (A) Real-time PCR analysis of the indicated neural markers in differentiating RUES2 cells. Control mock-transfected cells (NT, blue bars) and cells transfected twice with non-targeting siRNAs (siCo, red bars) or anti-NR2F2 siRNAs (siNR2F2, green bars) were collected for analysis at day 7 of differentiation as a monolayer. (B) Differentiating RUES2 cells were transfected twice with control non-targeting antisense LNA oligonucleotides (LNA-C; blue bars) or with anti-miR-302 LNA (LNA-302; red bars). Cells were collected at day 8 and levels of indicated neural markers were assessed by real-time PCR analysis. Asterisks in A and B indicate significant difference compared with the respective controls (Student's t-test; P<0.05). (C) Schematic representation of the ePiggyBac-based inducible system for NR2F2 ectopic expression. TRE, TET responsive promoter; pA, polyadenylation signal. Pubc, human Ubiquitin C promoter; BsdR, Blasticidin resistance gene. In the ePB-TET-on vector, the constitutive CAG promoter drives a fusion gene encoding for the doxycycline-activated TET protein fused to the hygromycin resistance protein through a self-cleavage T2A peptide. (D–K) Immunostaining analysis of RUES2 cells stably transfected with the NR2F2-inducible system and left untreated (D–G) or cultured in presence of doxycycline (H–K). Cells were stained for OCT4 (D, H), NR2F2 (E, I) and counterstained with Sytox Orange (F, J). The scale bar in D represents 100 μm and can be applied to all panels.