FIGURE 4.

Dose-dependent valproic acid treatment increases the endogenous content of α-synuclein, induces its redistribution to cytoplasmic foci, and affects mitochondrial Ca²⁺ transients and autophagic process. HeLa cells were incubated with VPA in DMEM at 37 °C in CO₂ atmosphere for 6 days at the indicated doses and then transfected with mtAEQ or mtRFP or mtGFP. Western blotting (A) and immunocytochemistry analysis (B) of α-syn expression levels and distribution after VPA treatment is shown. Numbers in panel A refer to normalized α-syn/β-actin ratio ± S.E. in four independent Western blottings. Statistical analysis (C) and representative experiments (D) of mitochondrial Ca²⁺ measurements in HeLa cells treated with VPA are shown. Results are the mean ± S.E. ***, p < 0.0001; **, p < 0.005; *, p < 0.01; NS, not significant. Where indicated the cells were stimulated with 100 μm histamine. The traces are representative of at least five independent experiments. E, mitochondrial morphology in HeLa cells treated with VPA was evaluated under fluorescent microscope by observing cotransfected mtRFP. F, immunocytochemistry analysis with anti α-syn antibody in HeLa cells treated with 1 mm VPA and transfected with mtGFP is shown. No α-syn and mtGFP colocalization was observed in these conditions. G, Western blotting analysis of LC3 I, LC3 II, and p62 levels in VPA-treated cells is shown; β-actin levels were also shown. Bars indicate the average values obtained by densitometric analysis of five independent experiments. Results are the mean ± S.E. **, p < 0.001.