Figure 1. Raf-Erk signal activation at high NSC densities induces cell proliferation and inhibition of neuronal differentiation.

(A–I), To obtain cultures at 50–80% cell confluence, NSCs from E14 rat embryonic cortices were plated at 3–4 × 10⁴/cm² in FGF2-supplemented N2 medium, and transduced with ca-Raf or control virus (viral titers: 10 × 10¹⁰ transduction unit, TU) on the following day. The cells were further cultured in N2 (without FGF2) for 4 days. Cell numbers on post-transduction days 0–4 (A–C), and numbers of cells positive for the proliferation markers Ki67 and M-phase pHH3 (D–F), and for the neuronal marker TUJ1 (G–I) were counted on post-transduction day 4. *p < 0.001, n = 3, Student’s t-test. Scale bar = 50 μm. (J–O), Dose-dependent ca-Raf effects on proliferation and neuronal differentiation. Levels of Erk activation were readily controlled by varying ca-Raf viral titers (J–M). Cortical NSC cultures (plated at 4 × 10⁴/cm²) were transduced with 0.5–10 × 10¹⁰ TU of ca-Raf virus, and stained with fluorescent pERK1/2 antibody 2 days later (J–L). Insets, high-magnification images of the boxed areas. Scale bar = 50 μm. Erk activation in individual cells was estimated from the mean fluorescence intensity (MFI) of individual pERK1/2 -stained cells using LAS image analysis (Leica). 54(2 × 10¹⁰ TU) and 63(10 × 10¹⁰ TU) stained cells were selected at random, and the margins of the individual cells were drawn. MFIs within the outlined area were calculated by comparison with the overall background intensity (L). *p < 0.001, Student’s t-test. Erk activation (pERK1/2 protein level) in cultures transduced with different titers of ca-Raf virus was further analyzed by western blotting (M). Dose-dependent Raf-Erk effects on cell proliferation and neuronal differentiation were estimated by counting total viable cells (N) and neurons positive for TUJ1 (O) in cultures 6 days after transduction. *p < 0.001, n = 3 cultures, one-way ANOVA.