Journal: Neuromolecular medicine
Article Title: Inhibitors of Histone Deacetylases Enhance Neurotoxicity of DNA Damage
Figure Lengend Snippet: The pro-apoptotic activation of p53 is enhanced by nonselective HDACis. a, b Neurons were treated with etoposide in the presence or absence of the pan-HDACis for 6 h as indicated (vehicle, 0.2 % DMSO, V; 0.1 μM TSA, TS; 10 μM SBHA, SB; 10 μM SAHA, SA). Levels of the Ser15-phosphorylated (P-p53) p53 and the total p53 (T-p53) were determined by immunoblotting; the membranes were reprobed for β-actin to ensure equal protein loading in each lane . a Blots from representative experiments; numbers under the blots indicate optical density ratios of the bands for the P-p53 and the β-actin or the T-p53 and the β-actin (fold control). b Quantification of phospho-p53 levels as determined in three independent experiments. Pan-HDACis increased p53 activation in response to etopo-side. In addition, TSA or SBHA weakly increased p53 phosphorylation/accumulation without etoposide. c Apoptosis in response to the overexpressed p53 is enhanced by the pan-HDACi TSA. Neurons were co-transfected with expression plasmids for β-gal and either a temperature-sensitive mutant of p53 (TS-p53) or an empty expression vector (pCMV, vector; 0.2 + 0.1 μg of plasmid DNAs/5 × 10 5 neurons, respectively). For next 24 h, neurons were kept at 37 °C. At this temperature, TS-p53 remained in an inactive conformation. Then, neurons were placed at 32 °C restoring wild-type p53 conformation of TS-p53. In addition, cells were treated with TSA as indicated. At 24 h after the temperature shift, TS-p53 induced apoptosis in TSA-treated neurons. Representative micrographs of transfected (i.e., β-gal-positive) neurons are shown; a surviving neuron with uniformly stained nucleus is indicated by an arrow ; an arrowhead identifies an apoptotic neuron with fragmentation of nuclear chromatin. The graph presents quantitation of the apoptosis response from three independent experiments (2 sister cultures/ experiment/condition). In b, c , error bars represent SD; NS p > 0.05; * p
Article Snippet: Immunofluorescence for β-gal was performed using a rabbit anti-β-gal antibody (MP Biomedicals) as described previously ( ).
Techniques: Activation Assay, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Staining, Quantitation Assay