KWV induces neuronal differentiation in the presence of EGF and FGF2.

<p>After expansion for 1 week in the presence of EGF and FGF2, NSCs were dissociated and plated. Then cells were treated with either DMSO or KWV (2.5 μM) in the presence of growth factors for 2 days and the mRNA expression levels of <i>notch1</i> (A) and <i>hes1</i> (B) were shown by RT PCR followed by real-time PCR. <i>Gapdh</i> was used as an internal control. Data were presented as mean ± SEM (n = 3). The <i>gfap</i> (C), <i>neuroD</i> (D), and <i>βIII tubulin</i> (E) mRNA expression levels of NSCs treated with DMSO or 2.5 μM KWV for 24 h in the presence of mitogens. Values were expressed as mean ± SD (C and D; n = 4) or mean ± SEM (E; n = 3). (F) The expression of <i>miR-9</i>, <i>miR-29a</i>, <i>miR-124</i>, and <i>miR-181a</i> in cells treated with DMSO (white bar) or 2.5 μM KWV (black bar) for 24 h in the presence of growth factors. <i>RNU6</i> was used as a loading control. The values were mean ± SD (n = 4). (G) Representative immunofluorescence images of cells treated with DMSO or 2.5 μM KWV for 43 h in the presence of EGF and FGF2. Cells were immunostained with TuJ1 antibody and nuclei were identified by DAPI staining [TuJ1-positive neurons (green), nuclei (blue)]. Scale bar, 50 μm. (H) Quantification of neurons. TuJ1-positive cells were counted and normalized to total cell number. Cell count data were presented as mean ± SD (n = 3). Statistical analysis of all data was performed using the Student’s <i>t</i>-test (*P < 0.05 and **P < 0.01 vs. control).