Figure 2. Treatment with VPA increases HSP70 protein, mRNA and activity levels, as well as histone acetylation levels in the HSP70 promoter in cortical neurons.
A. Rat cortical neurons were treated with vehicle or VPA (0.25-1.0 mM) for 72 hours starting at DIV-7. HSP70, acetylated histone H3 (AcH3), trimethylated at lysine 4 histone H3 (H3K4Me3), and β-actin protein levels were determined by immunoblotting. Quantified data of the blots on the right panel are mean ± SEM relative to control from three independent experiments. *p<0.05 compared with the control. B. Cortical neurons were treated with 1 mM VPA for the indicated periods of time and HSP70 and β-actin protein levels were determined on DIV-11 by immunoblotting. C. Cortical neurons at DIV-8 were treated with 1 mM VPA or vehicle for 24 h. Protein levels of HSP70 and HDAC1, which was used as a control for a nuclear protein, were determined in nuclear fractions by immunoblotting. D. Immunostaining of HSP70 (red) in cortical neurons after treatment with 1 mM VPA for 72 hours. DAPI nuclear staining shown in blue. Scale bar = 20μm. E. Rat cortical neurons were treated with 1 mM VPA for the indicated periods of time. Levels of the mRNA transcripts of the rat HSP70-1/HSP70-2 genes were determined by semi-quantitative RT-PCR on DIV-11. The right panel shows mean ± SEM of relative HSP70 mRNA values quantified from three independent experiments. *p<0.05. F, G. Rat cortical neurons and SH-SY5Y cells were transfected with human HSP70-1 or HSP70-2 promoter constructs. Neurons were treated with the indicated VPA concentrations for 24 or 72 hours, and HSP70-1 and HSP70-2 promoter activities were determined by dual luciferase assay on DIV-9. HSP70-1 and HSP70-2 promoter activities were determined in SH-SY5Y cells after 24 hours treatment with VPA by dual luciferase assay. Data are mean ± SEM from three to four independent experiments of values relative to control. *p<0.05; **p<0.01 compared with the control. H. Cortical neurons at DIV-7 were treated with 1 mM VPA or vehicle for 72 hours and chromatin was prepared and sheared by sonication. The protein/DNA complex was then incubated with antibody against acetylated histone H3 or without antibody (No AB) for ChIP analysis. Representative gel electrophoresis and representative fluorescence graph of PCR amplification of HSP70-2 gene promoter are shown.