AMPK activation via ROS can thus anticipate impending ATP depletion and prevent or limit this by down-regulating ATP dependent processes. qRT-PCR data depicted in Fig 3< 0.0001, *** < 0.001, ** < 0.01, * < 0.05.(TIF) pone.0134049.s005.tif (269K) GUID:?80DAC67B-9FEC-4005-A92A-FE3599E4AAAD S6 Fig: Isotope distribution. High-resolution dMS chromatogram (top) and mass spectrum (bottom) showing the isotope distribution for the tryptic peptide ATEMVEVGPEDDEVGAERGEATDLLR derived from Polymerase I and transcript release factor (Ptrf) with monoisotopic m/z = 930.103 Da and retention time 43.5 minutes. Colored lines show the average transmission for 4 WT (blue), 4 KO (reddish), 4 WT+Myc (green), 4 KO+Myc (pink), and 6 pooled control (tan) samples.(TIF) pone.0134049.s006.tif (430K) GUID:?FE95D29D-9066-4B7C-BB5C-A1D4AF6ED4A3 S7 Fig: Immuno-blotting for determined pyruvate metabolizing enzymes. Pyruvate dehydrogenase (PHDE) and Ser293 (activated) phosphorylated PDHE. Pyruvate dehydrogenase kinase (PDK1), Pyruvate DPCPX dehydrogenase phosphatase (PDP2), Pyruvate kinase M1 and M2 (PKM1/2), and -actin loading control.(TIF) pone.0134049.s007.tif (585K) GUID:?B32D6E1C-228A-432F-91E3-0781A29D42E5 S1 Table: qRT-PCR primers used in the current study. (DOCX) pone.0134049.s008.docx (19K) GUID:?E3D9D089-AB1E-42C5-AD55-4B2C473C76D4 S2 Table: Antibodies used in the current study. (DOCX) pone.0134049.s009.docx (14K) GUID:?85991627-A061-4F9A-BA19-05735EABD953 S3 Table: 345 mitochondrial proteins identified by LC-MS/MS analysis. Protein name, including the organism name (OS), gene name (GN), protein presence (PE, a numerical value describing the evidence of presence for the protein) and sequence version (SV). Gene name is usually how the protein is usually identified throughout the paper, followed by the primary accession number for reference. Overall p-value is usually calculated by a two way ANOVA. p- and q-values<0.05 are highlighted in red text throughout the table. The mean protein intensities are prepared and run in 4 individual samples for each cell type. Fold switch, p-value and false discovery rate (q-value) were calculated as explained in Statistical Analysis and the selected features are recognized by blue text in the fold switch columns. Features DPCPX were selected by a conservative cut off of q<0.05, with the exception of the comparison of AMPK WT to KO. KO proteins experienced an overall slightly higher average intensity, so to reduce potential bias, proteins with greater large quantity in KO but with fold switch less than 2.6 (twice the fold switch of overall mitochondrial large quantity in KO samples) were not considered.(XLSX) pone.0134049.s010.xlsx (625K) GUID:?CA43DA47-168F-4158-AEB6-3B107A861AF9 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes. Because DPCPX Myc over-expression depletes ATP with the resultant activation of Rabbit Polyclonal to Collagen V alpha1 AMPK, we explored the potential co-dependency of and cross-talk between these proteins by comparing the consequences of acute Myc induction in (WT) and (KO) murine embryo fibroblasts (MEFs). KO MEFs showed a higher basal rate of glycolysis than WT MEFs and an appropriate increase in response to activation of a Myc-estrogen receptor (MycER) fusion protein. However, KO MEFs experienced a diminished ability to increase Oxphos, mitochondrial mass and reactive oxygen species in response to MycER activation. Other differences between WT and KO MEFs, either in the basal state or following MycER induction, included abnormalities in electron transport chain function, levels of TCA cycle-related oxidoreductases and cytoplasmic and mitochondrial redox says. Transcriptional profiling of pathways relevant to glycolysis, Oxphos and mitochondrial structure and function also uncovered significant differences between WT and KO MEFs and their response to MycER activation. Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their constant state. Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, DPCPX which dictate pyruvate and acetyl coenzyme A large quantity, were also differentially responsive to Myc and AMPK and could account for some of DPCPX the differences in basal metabolite levels that were also detected by MS. Thus, Myc and AMPK are co-dependent and appearance to activate in significant highly.