Fig. 7

Effects of the tyrosine protein kinase activity inhibitor K252a on γ-Eno peptide-mediated cell survival and neurite outgrowth in SH-SY5Y cells. A, B SH-SY5Y cells were pretreated for 1 h with 50 nM to 500 nM K252a, the inhibitor of tyrosine protein kinase activity, followed by treatment with 100 nM γ-Eno peptide for 48 h. Quantification of cell survival using the MTS assay (A) and neurite outgrowth by counting the neurites (B), where cells with neurites longer than the cell diameter were scored. Data are means ± SD of three independent experiments (n = 3), each performed in quadruplicate (one-way ANOVA, Tukey’s test, *P < 0.05). C–E SH-SY5Y cells were treated with 100 nM γ-Eno peptide in the absence and presence of 200 nM K252a. C, D Representative images and quantification (as staining with 500 ng/mL TRITC-conjugated phalloidin) of fluorescence staining for actin filaments (solid arrows) by 500 ng/mL TRITC-conjugated phalloidin (red fluorescence) after 2 h of γ-Eno peptide treatment. Nuclei were counterstained with DAPI (blue fluorescence). Scale bars: 20 µm. Data are means ± SD of three independent experiments (n = 3), each performed in duplicate (one-way ANOVA, Tukey’s test, *P < 0.05). E Quantification of MAP2 expression after 24 h of γ-Eno peptide treatment by FACS analysis for immunostaining with a specific MAP2 antibody. Data are means ± SD of three independent experiments (n = 3), each performed in duplicate (one-way ANOVA, Tukey’s test, *P < 0.05). F, G SH-SY5Y cells were incubated for 1 h with inhibitor K252a (200 nM), followed by treatment with 100 nM γ-Eno peptide for additional 1 h. Representative Western blots of Akt (F), and ERK1/2 (G) activation. Total lysates (30 µg) were separated by SDS/PAGE and subjected to immunoblotting using the indicated antibodies. Graphs below indicate the relative values of phosphorylated forms compared to respective controls (total form of kinase) as means ± SD of two independent experiments (n = 2) (one-way ANOVA, Tukey’s test, *P < 0.05)