Effect of GATA-4 and USF-1 overexpression on PGC-1α promoter activity and mRNA expression.

<p>A. Representative western blots of protein extracts made from C₂C₁₂ cells transfected with either 2 or 4 µg of GATA-4 or USF-1 or an empty vector (EV) control. B. USF-1 and GATA-4 were co-transfected with the pGL3 (EV; 500ng) or the p851 PGC-1α promoter reporter construct (500ng) along with the appropriate empty vector controls. Relative luciferase activities were measured 48 hours after transfection and are plotted as the fold change above empty vector. Values are means±SEM, (n = 8); * p<0.05 versus p851-EV and #, p<0.05 versus p851-USF-1 or p851-GATA-4. C. Cells were transfected with 4 µg of USF-1 or an empty vector (EV) control. EtBr-stained DNA gel of PGC-1α amplified by PCR from EV- and USF-1 transfected cells. s12rRNA was used to verify equal loading. Data are representative of one experiment with conditions repeated in duplicate (AU = arbitrary units).</p

The human PGC-1α promoter.

<p>The nucleotide sequence +28 to −2190 corresponding to the proximal 2-kb hPGC-1α promoter is shown. The arrows indicate the transcription start sites, which have been previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003614#pone.0003614-Akimoto2" target="_blank">[14]</a>. Putative binding sites for transcription factors are either <i>underlined</i> or <i>overlined</i>. Also included are binding sites for transcription factors that have previously been characterized [12;13;19]. Numbers enclosed in circles represent the 5′- deletions of the PGC-1α promoter reporter constructs p 2215, p 1164, p 851, p501, p 191 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003614#pone-0003614-g002" target="_blank">Fig. 2</a>.</p

AMPK activation induces PGC-1α mRNA expression and transcriptionally activates the PGC-1α promoter.

<p>C₂C₁₂ cells were treated with either AICAR (1 mM) or Vehicle for 24 hrs. A. Representative Western Blot probed with a Phospho-AMPKα (Thr172), stripped and then re-probed with total AMPKα for loading control (upper panel). Summary of repeated experiments of the effect of AICAR on AMPK activation is shown (lower panel; n = 4). B. upper panel, EtBr-stained DNA gel of PGC-1α amplified by PCR from vehicle- and AICAR-treated cells. GAPDH was also amplified by PCR and used to verify equal loading. Lower panel: A summary of repeated experiments of the effects of AICAR on PGC-1α mRNA expression (n = 3). C. AICAR-induced transcriptional regulation of the PGC-1α promoter. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown (n = 4–6). D. The AICAR-responsive region (ARR) from −473 to −821 was cloned into the pGL4.23 minimal promoter vector and AICAR-induced transcriptional regulation of this region was assessed (n = 3). For all data, values are means±S.E.M, *, p<0.05 versus Vehicle-treated control; §, p<005 versus pGL4.23.</p

AMPK activation increases GATA/EBox DNA binding.

<p>A. Representative EMSAs of nuclear extracts from Vehicle− (−AIC) and AICAR− (+AIC) treated cells that were incubated with radiolabeled oligonucleotides corresponding to the EBox, SRE and IRS sequences found within the AICAR-responsive region of the PGC-1α promoter (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003614#pone-0003614-g001" target="_blank">Fig. 1</a>). B. A representative EMSA (<i>Left panel)</i> and a summary of multiple experiments (<i>Right panel)</i> showing the effect of AICAR (A) on GATA/EBox-DNA binding. Values are represented as means±S.E.M (n = 8) relative to vehicle-treated (V) cells. C. Representative EMSAs of nuclear extracts that were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt. Vehicle-treated cells were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt as well as antibodies against MyoD, USF-1 and c-Myc which are known to bind to the EBox sequence. FP: free probe, 25×, 50×, 100× CO: 25-fold, 50-fold, 100-fold molar excess of cold oligo, No Ab: No Antibody, SRF: SRF antibody, FKHR: Forkhead antibody, GATA-4: GATA-4 antibody, c-Myc: c-Myc Antibody, USF-1: USF-1 antibody. **The representative blot of IRS/DNA binding was made from parts of the same gel. D. Representative chromatin immunoprecipitation from cells treated with or without 1 mM AICAR for 24 hours. Protein/DNA complexes were immunoprecipitated with USF-1 antibody, or with non-specific IgG. Primers encompassing the region between −473 and −823 were used to analyze USF-1 binding to the PGC-1α promoter. The representative blot on the left was made from parts of the same gel. At right is a graphical summary of repeated experiments. Values are representative of means±S.E.M (n = 6).</p

DJ-1 and oxidative stress modulate PON2 activity.

<p>(<b>A</b>) Cultured WT and DJ-1 KO cortical neurons were treated with MPP⁺ (20 µM) for 12 hours and cells were washed and membrane was extracted. Crude membrane was exposed to the substrate C12 for 60 minutes and the percentage of remaining C12 was measured. (<b>B</b>) Cultured WT and DJ-1 KO cortical neurons were treated with MPP⁺ (20 µM) for 24 hours. Neurons were then exposed to DHC for 10 minutes and the amount of hydrolysis of DHC was assessed with measuring UV absorbance. One unit of PON2 activity is equal to 1 µmol DHC hydrolyzed<b>/</b>ml<b>/</b>min. (<b>C</b>) WT and DJ-1 KO MEFs were treated with hydrogen peroxide (100 µM) for 24 hours and PON2 activity was measured as described in B. (<b>D</b>) WT and DJ-1 KO MEFs were infected with adenovirus expressing DJ-1 or GFP alone as control. After 48 hours of expression, cells were lysed and exposed to C12 as the substrate for 60 minutes. Percentage of C12 remaining in activity buffer was measured. Statistical significance was assessed by Anova and post-hoc test Tukey on data obtained from three independent experiments (n = 3). * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001.</p

PON2 protects neurons against MPP^<b>+</b>.

<p>(<b>A</b>) Primary cortical neurons obtained from PON2 deficient or wild type mice were subjected to 10, 20 and 40 µM MPP⁺ treatment for 48 hours. Cells were lysed and viability was assessed by direct microscopy and counting intact nuclei. (<b>B</b>) WT and PON2 def cortical neurons were transfected with plasmid expressing Myc-PON2 and GFP (under independent promoters), or GFP as control, and subjected to 20 µM MPP⁺ for 48 hours. Cells were fixed and the nuclei were stained with Hoechst. Survival percentage represents the ratio of GFP-expressing cells with morphologically intact nuclei (D, a and b) to the total number of GFP positive cells. (<b>C</b>) WT and DJ-1 KO cortical neurons over-expressing PON2 and GFP or GFP alone as control (using adenovirus expressing PON2 or GFP) were subjected to 20 µM MPP⁺ for 48 hours. The survival assay was performed as described in part B. (<b>D</b>) Representative image of GFP positive neurons (a and c), and Hoechst-stained surviving (b) and dead (d) nuclei. (<b>E</b>) Western blot analysis of PON2 levels in PON2 deficient (PON2 def) and WT MEFs and also in WT MEFs infected with PON2-expressing adenovirus (WT+PON2 AV). The membrane was probed with PON2 antibody. (<b>F</b>) Western blot analysis for Myc in WT MEFs expressing control (Ctr) or Myc-PON2 plasmids. The Western blot was analyzed by Myc antibody. Statistical significance was assessed by Anova and post-hoc test Tukey on data obtained from three independent experiments (n = 3). * denotes p<0.05, **denotes p<0.01 and *** denotes p<0.001.</p