FIGURE 7.

HDAC2 mediates the role of nNOS in regulating the fate of adult NSCs. (A) Identification of cultured adult NSCs. Single-cell suspensions were seeded on polyornithine/laminin-coated coverslips, cultured as a monolayer for 24 h, and then fixed for nestin staining. At least 92% cells were nestin⁺ NSCs. In addition, cells were monolayer-cultured in the presence of 10 μM BrdU for 24 h and fixed for stain, and most cells were BrdU⁺ labeled. These cells could differentiate into β-III-Tubulin⁺ neurons and GFAP⁺ astrocytes after differentiation for 4 days. (B) Monolayer-cultured adult NSCs treated with 100 μM L-VNIO during the later 2 days of 4-day differentiation exhibit a marked decrease of neuronal differentiation. Immunoblots showing HDAC2 levels (C) and bar graph showing HDAC2 activity (D) in cultures treated with 100 μM L-VNIO or vehicle for the first 24 h during differentiation. (E,F) HDAC2 down-regulation rescues L-VNIO-induced neuronal differentiation reduction. 100 μM L-VNIO or vehicle was treated for the later 2 days during the 4-day differentiation of LV-HDAC2 shRNA- or LV-Control shRNA-infected adult NSCs. (E) Bar graph showing the percentage of newborn neurons. (F) Representatives of β-III-Tubulin⁺ neurons. Scale bars = 50 μm. Data are mean ± SEM (n = 3); ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. BrdU, bromodeoxyuridine; GAPDH, glyceraldehyde phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; HDAC2, histone deacetylase 2; LV-Control shRNA, lentiviral vector containing control shRNA; LV-HDAC2 shRNA, lentiviral vector containing shRNA of HDAC2; L-VNIO, N⁵-(1-imino-3-butenyl)-L-ornithine.