Effect of VPA on activities of ERK pathway components and on expression of p21Cip/WAF1 and Tuj1. NPCs grown in 10 ng/ml bFGF were treated with 1 mM VPA for 48 h. (A) Whole-cell lysates were subjected to immunoblotting for analysis of the presence of p-ERK, p-MEK, p-Raf-1, p21Cip/WAF1, Tuj1, or β-actin. Alternatively, NPCs were processed for immunofluorescent labeling of (B) p21Cip/WAF1 (green), (C) BrdU (red), or (D) Tuj1 (green). Nuclei were counterstained with DAPI (blue). All images are 200× magnifications. The percentage of cells that exhibited nuclear p21Cip/WAF1 in the presence and absence of VPA is presented, along with the percentage of BrdU-positive and Tuj1-positive cells. Error bars indicate the standard deviations of three independent experiments. Data represent mean ± SD of three separate experiments. P*** < 0.001, P** < 0.01 by Student's t-test. (B-C). Effects of inhibition of ERK pathway on p21Cip/WAF1 and Tuj1 induction by VPA. (E) NPCs grown in the presence of 10 ng/ml bFGF were treated with different combinations of 20 μM PD98059 and 1 mM VPA. (F) NPCs grown in the presence of 10 ng/ml bFGF were treated as in (E). In three independent experiments, cells within the 230-μm square were counted and expressed as a percentage of the total number of cells. Values represent the mean ± SD of three separate experiments. P** < 0.01, P*** < 0.001 by one-way ANOVA followed by Newman-Keuls test.