Endogenous products of APP metabolism negatively affect HIPK2 DNA-binding activity.

<p>(<b>a</b>) ChIP experiments were performed with anti-HIPK2 antibody on HEK-293 and HEK-APP cells that were also treated with β-secretase inhibitor at 1 µmol/L for 48 h and on HEK-293 treated with conditioned medium from HEK-APP cells for 48 h in the absence or presence of β-secretase inhibitor; PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for the human HIF-1α promoter as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010171#pone-0010171-g001" target="_blank">Figure 1</a>. (<b>b</b>) ChIP experiments were performed with anti-HIPK2 antibody on HEK-293 and HEK-APP cells that were also treated with β-secretase inhibitor at 1 µmol/L for 4 8h and on HEK-293 treated with conditioned medium from HEK-APP cells for 48 h in the absence or presence of β-secretase inhibitor; PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for the MT2A promoter. (<b>c</b>) HEK-293 and HEK-APP cells were transfected with MT2A-luc and HIF-1α-luc reporter construct and luciferase activity was measured 36 h after transfection. Results normalized to β-galactosidase activity are presented as fold of induction of luciferase activity ±S.D. At least three independent experiments performed in duplicate. * <i>p</i><0.01 (Student <i>t</i>-test). (<b>d</b>) MT2A and HIF-1α mRNA expression was determined in HEK-APP compared to HEK-293 cells by reverse-transcriptase (RT)-PCR. GAPDH was used as loading control.</p

Levels of Aβ 1-40 and Aβ 1-42 after treatment with β secretase inhibitor.

<p>Levels of Aβ 1-40 and Aβ 1-42 peptides were measured with a commercial ELISA kit in the cellular extracts and conditioned media of HEK-APP cells untreated or treated with β secretase inhibitor at 1 µmol/L for 48 hours. Results are representative of at least three independent experiments ± S.E.M.</p><p>* p<0.001 βSI treatment vs corresponding control.</p><p># p<0.01 βSI treatment vs corresponding control.</p

Zinc supplementation to HEK-APP restores p53 pro-apoptotic transcriptional activity.

<p>(<b>a</b>) HEK-293 and HEK-APP cells were transfected with p53AIP1-luc reporter construct and 24 h after transfection treated with doxorubicin (3.4 µM) and zinc (100 µM) for 24 h before luciferase activity was assayed. Results normalized to β-galactosidase activity are shown as relative luciferase activity ±S.D. At least three independent experiments performed in duplicate. * <i>p</i><0.05 vs HEK-293 or HEK-APP; ** <i>p</i><0.01 vs HEK-APP (Bonferroni Multiple Comparison test). (<b>b</b>) Bax mRNA expression was determined in HEK-APP compared to HEK-293 cells by reverse-transcriptase (RT)-PCR after treatment with doxorubicin (3.4 µM) and zinc (100 µM) for 24 h. GAPDH was used as loading control. (<b>c</b>) Total cell extracts of HEK-APP cells treated with doxorubicin (3.4 µM) and zinc (100 µM) for 24 h were analysed for Bax and p53 expression. Protein loading control was shown as Ponceau staining.</p

(A)correlative analysis between SOD activity and unfolded p53 on all samples (control, EOSAD and FAD) considered in this study.

<p>The equation of linear regression is y = −5,518x+0,1973 r² 0,2321 p = 0,01. (<b>B</b>) lymphocytes derived from a healthy subject were exposed to SOD inhibitor DETC (5 Mm) and 24 h later they were processed for SOD enzyme activity and the expression of PAb 240 -positive p53 isoform by using ELISA assay. Data are expressed as mean ± SEM of three different experiments, performed in triplicate. Statistical analysis was performed with t test with * p<0,001, ** p<0,001 vs untreated samples.</p

The enzymatic activity of glutathione peroxidase (GPX) and glutathione reductase (GRD).

<p>A) glutathione peroxidase (GPX) was measured in controls (n = 9), EOSAD (n = 9) and ADmut (n = 9) lymphocytes using specific assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029789#s4" target="_blank">method</a> section). B) glutathione reductase (GRD) was measured in controls (n = 9), EOSAD (n = 9) and ADmut (n = 9) lymphocytes using specific assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029789#s4" target="_blank">method</a> section).</p

Expression of SOD1 and SOD2 protein levels and activity in ADmut, EOSAD and control lymphocytes.

<p>Protein extracts derived from immortalized lymphocytes of ADmut, EOSAD and healthy individuals were prepared as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029789#s4" target="_blank">method</a> section. <b>A</b>) Western blot analysis carried out with monoclonal anti-SOD1 and anti-SOD2 antibodies on protein extracts derived from 2 controls, 2 ADmut, and 2 EOSAD. Tubulin expression was used to normalize the samples. <b>B</b>) and <b>C</b>) SOD1 and SOD2 levels of 9 controls, 9 ADmut and 9 EOSAD were measured using Scion Image program: quantitative analysis was expressed as intensity (optical density) of SOD1 or SOD2 bands over tubulin levels. <b>D</b>) enzymatic activity of superoxide dismutase (SOD) measured in controls (n = 9), EOSAD (n = 9) and ADmut (n = 9) lymphocytes using specific enzymatic assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029789#s4" target="_blank">method</a> section).</p

Demographic and clinical characteristic of the subjects enrolled in the study.

<p>Abbreviations: EOSAD, early onset sporadic AD; ADmut, familial AD; CTR, control; M, male; F, female; LOI, length of illness; MMSE, Mini Mental State Examination; N, number of individuals.</p><p>Values are expressed as mean ± SD.</p

Effects of peroxynitrite compound SIN-1 on p53 conformation.

<p>Lymphocytes derive from a healthy subject were exposed to 500 µM SIN-1 in the presence or absence of uric acid at the same concentration. In particular SIN-1 was added 30 min after uric acid addition and incubated for the next 4 hours. Cells were then processed for (<b>A</b>) RNS generation study by FACS analysis measuring DCF fluorescence; (<b>B</b>) Pab 240 positive p53 isoform (unfolded p53) measured by ELISA assay, and (<b>C</b>) the degree of p53 nitration on tyrosine residues investigated by immunoprecipitation experiment with the two conformational specific antibodies (PAb 1620 and pab 240) followed by immunoblottin, with anti-rabbit-anti-3NT or anti-goat anti-p53 (R19).</p

Oxidative profile in EOSAD, ADmut and control lymphocytes.

<p>Dot blot analysis on protein extracts derived from controls (n = 9), EOSAD (n = 9) and ADmut (n = 9) lymphocytes were performed using specific antibodies against oxidative stress markers: protein-bound HNE (<b>A</b>), 3NT (<b>B</b>) and PC (<b>C</b>). Tubulin expression was used to normalize the samples. * p<0,05 control vs ADmut.</p