Journal: Cell Death & Disease

Article Title: Mitochondrial complex II is a source of the reserve respiratory capacity that is regulated by metabolic sensors and promotes cell survival

doi: 10.1038/cddis.2015.202

Figure Lengend Snippet: The RRC is regulated by pyruvate dehydrogenase kinases. ( a – c ) Neonatal rat cardiac myocytes were incubated for 24 h in complete growth medium under normoxic (atmospheric O 2 ) ( a ) or hypoxic (<1% O 2 ) ( b and c ) conditions. After this period, RNA was either immediately extracted and subjected to qPCR for the indicated genes ( b ), or the medium was replaced with one that is glucose and fatty acid free, or one containing 17.5 mM glucose, 100 μ M palmitate-BSA, or glucose+palmitate-BSA, as indicated, in normoxic conditions, for another 24 h ( a and c ). The results were averaged plotted as relative values to those from cells incubated in base medium in normoxic conditions adjusted to 1 ( a and c ) or control normoxia adjusted to 1 ( b ), n =3. Error bars represent standard error of the mean (S.E.M.), * P <0.05 versus control. ( d ) Neonatal cardiac myocyte was incubated for 24 h in complete growth medium and then infected with a control or adenoviruses (Ad) harboring PDK1, PDK4 or Hif-1 α , for 24 h. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose plus 100 μ M palmitate for 1 h. The mitochondrial stress test was then performed as described in Materials and Methods, n =4–6, each experiment was performed twice. Error bars represent S.E.M., * P <0.05 max OCR versus basal OCR for control at the time point indicated; # P <0.05 max OCR for control versus max OCR of Pdk1 or Hif-1 α treated. ( e ) Neonatal cardiac myocytes were cultured for 24 h in complete growth medium containing vehicle or 1 μ M etomoxir, a Cpt1 inhibitor. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose plus 100 μ M palmitate containing vehicle or 1 μ M etomoxir, for 1 h. The mitochondrial stress test was then performed as described in Materials and Methods, n =4–6, each experiment was performed twice. Error bars represent S.E.M., * P <0.05 max OCR versus basal OCR for control at the time point indicated; # P <0.05 max OCR for control versus max OCR for etomoxir treated, at the time point indicated ( f and g ). Neonatal rat cardiac myocytes were cultured in complete growth medium containing vehicle or 1 mM DCA and either remained in normoxic conditions (atmospheric O 2 ) ( f ) or were exposed to hypoxia (<1% O 2 ) ( g ), for 24 h. At the end of this period, the medium was changed to base medium containing 17.5 mM glucose or 17.5 mM glucose plus 100 μ M palmitate-BSA containing vehicle or DCA, as indicated, for an additional 24 h. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose or 17.5 mM glucose plus 100 μ M palmitate-BSA containing vehicle or 1 mM DCA, for 1 h. The mitochondrial stress test was then performed as described in Materials and Methods, n =4–6, each experiment was performed three times. Error bars represent S.E.M., * P <0.05 max OCR versus basal OCR for DCA-treated cells, at the time point indicated; # P <0.05 max OCR for DCA-treated versus max OCR for untreated, at the time point indicated

Article Snippet: The human cDNA clones for Pdk1 (SC321678/NM_002610), Pdk4 (SC118542/NM_002612), Sdhaf1 (NM_001042631) and Sirt3 (NM_012239) were purchased (Origene Technologies, Inc., Rockville, MD, USA) and cloned into recombinant adenovirus vectors, propagated, and titered as previously described by Graham and Prevec.

Techniques: Incubation, Control, Infection, Cell Culture

Journal: Molecular Cancer Therapeutics

Article Title: Discovery of a novel class of AKT pleckstrin homology domain inhibitors

doi: 10.1158/1535-7163.mct-07-2276

Figure Lengend Snippet: Figure 1. Interactions of compound 1 with the human AKT1 PH domain. A, schematic representation of the interaction of compound 1 (NSC 348900, 4-[1-(4-chlorobenzoyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-ylazo]-N-pyrimidine-2-yl-benzenesulfonamide, C21H16ClN7O4S) with amino acid resi- dues of the AKT1 PH domain (Arg86, Asn53, Arg23, and Ile19). B, the docking pose of compound 1 with the AKT1 PH domain. The AKT1 PH domain (red); residues Arg23, Arg25, and Arg86 are colored according to atom type. Compound 1 is colored according to atom type (capped stick). Hydrogen bonding interactions (dotted lines). The sulfamido group interacts with Arg86 through a hydrogen bonding interaction whereas similar hydrogen bonds are involved in the interaction of the diazopyrazolyl group with Arg23. These two arginines are intimately involved in the interaction with the phosphate head groups of the substrate phosphoinositol-1,3,4,5-tetrakisphosphate. C and D, the binding mode of compound 1 in the binding pocket of the PH domain of PDK1 with the amino acids involved in the binding pocket (D). Note that compound 1 exhibits the reverse binding pose in the PDK PH domain similar to compound 2 in the PH domain of AKT1. Supplementary Fig. 1A and B show the binding mode of compound 1 in the binding pocket of the PH domain of IRS1.

Article Snippet: Expression and Purification of Recombinant PH Domains Recombinant mouse AKT1 PH domain amino acids 1 to 111 (UBI/Millipore), human AKT1 PH domain amino acids 1 to 111 (Origene, NM005163.2), human IRS1 PH domain amino acids 12 to 133 (Invitrogen), and human PDK1 PH domain amino acids 407 to 549 (Origene, NM002613.3) were cloned by PCR into EcoRI/XhoI sites in pGEX-4T1 inducible bacterial expression plasmid (GeneStorm, InVitrogen) transformed into BL21(DE3) Escherichia coli .

Techniques: Binding Assay