Fig. 4. Inhibitory activity of the atonal-related bHLH proteins on Nurr1-induced expression of DA neuronal phenotype. The cortical precursor cells, at day 3 of in vitro expansion, were transduced with Nurr1 plus Ngn1, Ngn2, NeuroD or LacZ (control) using retroviral infection. After 3 days of differentiation, the cultures were subjected to immunocytochemical (A-E), immunoblot analyses (F) for TH and RT-PCR analyses (G) for TH, AADC (DA synthesis), VMAT2 (DA storage) and DAT (DA uptake). (A-D) Representative images of TH/TuJ1 immunocytochemistry on the cells transduced with LacZ (A), Ngn1 (B), Ngn2 (C), and NeuroD (D) along with Nurr1. Note that TH⁺ cell numbers were reduced by the bHLH co-expression, with increases of TuJ1⁺ neurons. Insets, DAPI nuclear staining of the same fields. The graph in E represents percentage of TH⁺ cells out of total (DAPI⁺) cells. ^*Significantly different from the control at P<0.001. Bars, 10 µm.