Fig. 7. Rescue of neuronal maturation in mutant cells by lentiviral Sox2 expression at early stages of in vitro differentiation. (A) Immunofluorescence for Sox2 (red) (R&D) and GFP (green), encoded by Sox2-IRES-GFP lentivirus, in cells infected at day 1 (d1) or day 4 (d4), compared with non-infected (ni) control. Immunofluorescences were performed the day after infection. Efficient infection (high proportion of GFP-positive cells) is coupled to clear Sox2 overexpression, which is observed at variable levels in transduced cells. (B) β-tubulin- and GFP immunofluorescence, at differentiation day 9, of mutant cells transduced with Sox2-GFP lentivirus at day 1 (d1), or day 4 (d4), compared with non-infected (ni) control, or the control infected with GFP-only transducing virus. Abundant well-arborized β-tubulin-positive cells (arrowheads indicate two of them) are observed in cultures transduced at day 1 with the Sox2-expressing virus, but not in cells transduced at day 4, or in controls. (C) GFP (green) and β-tubulin (red, top) or MAP2 (red, bottom) immunofluorescence shows that well-arborized neuronal cells (arrowheads) are always double-positive for the neuronal marker and for GFP, indicating that they derive from a Sox2-transduced cell. By contrast, some poorly developed neuronal cells (arrow) are not green, thus presumably originating from non-transduced cells. (D) Fold-increase in numbers of MAP2-positive and well-arborized β-tubulin-positive cells in mutant cells infected with Sox2-lentivirus at differentiation day 1, when compared with infection at day 4, or with control virus (day 1) expressing GFP but not Sox2. Values represent fold increase in numbers of MAP2-positive or well-arborized β-tubulin-positive cells (arrowheads in B,C for examples) relative to non-infected control. In day 1 transduced cells, numbers of well-arborized β-tubulin-positive and of MAP2-positive cells were 3.7% and 4.3%, respectively. In a parallel experiment using wild-type control cells mock-treated in the same way with a non-Sox2-expressing virus, the corresponding values were 5.7 and 6.2%. Data from two experiments in duplicate.