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    Thermo Fisher stepone real time polymerase chain reaction system
    Expression of PPAR factors in MTO1 and GTPBP3 defective cells. ( A) <t>qRT-PCR</t> analysis of the PPARα, β/δ and γ <t>mRNA</t> expression in MTO1 human fibroblasts (MTO1 HF). Data are represented as fold change respect to WT HF values ( B,C and D ) Representative immunoblots of PPARγ in WT and MTO1 HF ( B ), in MTO1 siRNA1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells ( C ), and in GTPBP3 siRNA1-, GTPBP3 siRNA 2- and NC siRNA-transfected 143B cells ( D ). Scatter plots show the densitometric analysis of PPARγ normalized to porin and represented as fold change relative to control cells. All data are the mean ± SD of at least three different experiments. Differences from control values were found to be statistically significant at *p
    Stepone Real Time Polymerase Chain Reaction System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 32983 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of PPAR factors in MTO1 and GTPBP3 defective cells. ( A) qRT-PCR analysis of the PPARα, β/δ and γ mRNA expression in MTO1 human fibroblasts (MTO1 HF). Data are represented as fold change respect to WT HF values ( B,C and D ) Representative immunoblots of PPARγ in WT and MTO1 HF ( B ), in MTO1 siRNA1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells ( C ), and in GTPBP3 siRNA1-, GTPBP3 siRNA 2- and NC siRNA-transfected 143B cells ( D ). Scatter plots show the densitometric analysis of PPARγ normalized to porin and represented as fold change relative to control cells. All data are the mean ± SD of at least three different experiments. Differences from control values were found to be statistically significant at *p

    Journal: Scientific Reports

    Article Title: Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis

    doi: 10.1038/s41598-018-19587-5

    Figure Lengend Snippet: Expression of PPAR factors in MTO1 and GTPBP3 defective cells. ( A) qRT-PCR analysis of the PPARα, β/δ and γ mRNA expression in MTO1 human fibroblasts (MTO1 HF). Data are represented as fold change respect to WT HF values ( B,C and D ) Representative immunoblots of PPARγ in WT and MTO1 HF ( B ), in MTO1 siRNA1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells ( C ), and in GTPBP3 siRNA1-, GTPBP3 siRNA 2- and NC siRNA-transfected 143B cells ( D ). Scatter plots show the densitometric analysis of PPARγ normalized to porin and represented as fold change relative to control cells. All data are the mean ± SD of at least three different experiments. Differences from control values were found to be statistically significant at *p

    Article Snippet: For mRNA quantification, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

    MTO1-defective cells exhibit altered expression of metabolism genes and differ from GTPBP3-defective cells in the bioenergetics profile. ( A ) qRT-PCR analysis of mRNA expression of genes related to glycolysis ( GLUT1: glucose transporter 1, PKF1: phosphofructokinase, LDHA and LDHB: lactate dehydrogenase A and B, respectively, PDK4: pyruvate dehydrogenase kinase 4 and MPC1: mitochondrial pyruvate carrier 1 ); glutaminolysis ( ASCT2: glutamine/amino acid transporter 2, SN2: glutamine/amino acid transporter system N , and GLS: glutaminase ); cellular fatty acid (FA) uptake ( FAT/CD36: fatty acid translocase , and FABP3: fatty acid binding protein 3 ); mitochondrial fatty acid (FA) uptake ( CPT1a and CPT1b: carnitine palmitoyltransferase I a and b ); fatty acid oxidation ( LCAD: long-chain acyl-CoA dehydrogenase, MCAD:medium-chain acyl-CoA dehydrogenase , and HADH: hydroxyacyl-CoA dehydrogenase ); fatty acid synthesis ( ACC: Acetyl-CoA carboxylase and FAS: fatty acid synthase ), in MTO1 (MTO1 HF) human fibroblasts. Data are expressed as fold change respect to WT HF and shown in a heatmap. The colour and the corresponding value in log2 scale are plotted on the right. ( B ) Oxygen consumption rates of digitonin-permeabilized cells in the presence of 5 mM ADP, 0.2 mM octanoyl-carnitine (Oct) and malate ( M ), which was added at three different concentrations (0.05, 0.1 and 2 mM). ( C ) Oxygen consumption rates of digitonin-permeabilized cells in the presence of 5 mM ADP, 0.2 mM octanoyl-carnitine (Oct), 2 mM malate ( M ) and after sequential addition of substrates for Complex I (5 mM pyruvate ( P ) and 10 mM glutamate ( G ); PGMOctp) and Complex II (succinate ( S ); PGMSOctp), the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (CCCP, stepwise titration in 0.05 μM increments; PGMSOcte), and the Complex I inhibitor (0.5 μM rotenone; Se). Oxygen consumption rates (OCRs), expressed as picomoles (pmol) per second (s) per million of cells (Mill), were normalized to the mitochondrial copy number (mtDNA/nDNA ratio) in each sample. p denotes phosphorylation of ADP + Pi to ATP. e denotes electron transfer system (ETS) capacity at optimum CCCP concentration (noncoupled respiration). Data represent the means ± SD from at least 3 independent determinations. Differences from wild-type (WT) or Negative Control (NC) values were found to be statistically significant at *p

    Journal: Scientific Reports

    Article Title: Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis

    doi: 10.1038/s41598-018-19587-5

    Figure Lengend Snippet: MTO1-defective cells exhibit altered expression of metabolism genes and differ from GTPBP3-defective cells in the bioenergetics profile. ( A ) qRT-PCR analysis of mRNA expression of genes related to glycolysis ( GLUT1: glucose transporter 1, PKF1: phosphofructokinase, LDHA and LDHB: lactate dehydrogenase A and B, respectively, PDK4: pyruvate dehydrogenase kinase 4 and MPC1: mitochondrial pyruvate carrier 1 ); glutaminolysis ( ASCT2: glutamine/amino acid transporter 2, SN2: glutamine/amino acid transporter system N , and GLS: glutaminase ); cellular fatty acid (FA) uptake ( FAT/CD36: fatty acid translocase , and FABP3: fatty acid binding protein 3 ); mitochondrial fatty acid (FA) uptake ( CPT1a and CPT1b: carnitine palmitoyltransferase I a and b ); fatty acid oxidation ( LCAD: long-chain acyl-CoA dehydrogenase, MCAD:medium-chain acyl-CoA dehydrogenase , and HADH: hydroxyacyl-CoA dehydrogenase ); fatty acid synthesis ( ACC: Acetyl-CoA carboxylase and FAS: fatty acid synthase ), in MTO1 (MTO1 HF) human fibroblasts. Data are expressed as fold change respect to WT HF and shown in a heatmap. The colour and the corresponding value in log2 scale are plotted on the right. ( B ) Oxygen consumption rates of digitonin-permeabilized cells in the presence of 5 mM ADP, 0.2 mM octanoyl-carnitine (Oct) and malate ( M ), which was added at three different concentrations (0.05, 0.1 and 2 mM). ( C ) Oxygen consumption rates of digitonin-permeabilized cells in the presence of 5 mM ADP, 0.2 mM octanoyl-carnitine (Oct), 2 mM malate ( M ) and after sequential addition of substrates for Complex I (5 mM pyruvate ( P ) and 10 mM glutamate ( G ); PGMOctp) and Complex II (succinate ( S ); PGMSOctp), the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (CCCP, stepwise titration in 0.05 μM increments; PGMSOcte), and the Complex I inhibitor (0.5 μM rotenone; Se). Oxygen consumption rates (OCRs), expressed as picomoles (pmol) per second (s) per million of cells (Mill), were normalized to the mitochondrial copy number (mtDNA/nDNA ratio) in each sample. p denotes phosphorylation of ADP + Pi to ATP. e denotes electron transfer system (ETS) capacity at optimum CCCP concentration (noncoupled respiration). Data represent the means ± SD from at least 3 independent determinations. Differences from wild-type (WT) or Negative Control (NC) values were found to be statistically significant at *p

    Article Snippet: For mRNA quantification, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Titration, Concentration Assay, Negative Control

    MTO1 and GTPBP3 defects produce opposite effects on the AMPK/UCP2 axis. (A,E and I) Representative immunoblots of phosphor-Thr172-AMPKα and UCP2 in wild-type (WT HF) and MTO1 (MTO1 HF) human fibroblasts ( A ), in MTO1 siRNA1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells ( E ), and in GTPBP3 siRNA1-, GTPBP3 siRNA 2- and NC siRNA-transfected 143B cells ( I ). (B,F and J) Densitometric analysis of phospho-Thr172-AMPKα normalized to AMPKα and represented as fold change relative to control cells. (C,G and K) Densitometric analysis of UCP2 normalized to porin and represented as fold change relative to control cells. (D,H and L) qRT-PCR analysis of the UCP2 mRNA expression in WT and MTO1 HF ( D ), in MTO1 siRNA1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells ( H ), and in GTPBP3 siRNA1-, GTPBP3 siRNA 2- and NC siRNA-transfected 143B cells ( L ). All data are the mean ± SD of at least three different experiments. Differences from control values were found to be statistically significant at *p

    Journal: Scientific Reports

    Article Title: Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis

    doi: 10.1038/s41598-018-19587-5

    Figure Lengend Snippet: MTO1 and GTPBP3 defects produce opposite effects on the AMPK/UCP2 axis. (A,E and I) Representative immunoblots of phosphor-Thr172-AMPKα and UCP2 in wild-type (WT HF) and MTO1 (MTO1 HF) human fibroblasts ( A ), in MTO1 siRNA1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells ( E ), and in GTPBP3 siRNA1-, GTPBP3 siRNA 2- and NC siRNA-transfected 143B cells ( I ). (B,F and J) Densitometric analysis of phospho-Thr172-AMPKα normalized to AMPKα and represented as fold change relative to control cells. (C,G and K) Densitometric analysis of UCP2 normalized to porin and represented as fold change relative to control cells. (D,H and L) qRT-PCR analysis of the UCP2 mRNA expression in WT and MTO1 HF ( D ), in MTO1 siRNA1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells ( H ), and in GTPBP3 siRNA1-, GTPBP3 siRNA 2- and NC siRNA-transfected 143B cells ( L ). All data are the mean ± SD of at least three different experiments. Differences from control values were found to be statistically significant at *p

    Article Snippet: For mRNA quantification, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Western Blot, Transfection, Quantitative RT-PCR, Expressing

    Regulation of UCP2 is AMPK- and PPARγ-dependent in MTO1 fibroblasts. (A) qRT-PCR analysis of the UCP2 mRNA expression in MTO1 human fibroblasts (MTO1 HF), treated or not with 5 μM rosiglitazone (RGZ) for 1 h. Data are represented as fold change respect to WT HF values. (B) ). The scatter plot shows densitometric data for UCP2 normalized to porin and phosphor-Thr172-AMPKα normalized to AMPKα, and represented as fold change relative to WT HF. (C) ). The scatter plot shows densitometric data for UCP2 normalized to porin and phosphor-Thr172-AMPKα normalized to AMPKα, and represented as fold change relative to WT HF. All data are the mean ± SD of at least three different experiments. Differences from WT HF values were found to be statistically significant at *p

    Journal: Scientific Reports

    Article Title: Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis

    doi: 10.1038/s41598-018-19587-5

    Figure Lengend Snippet: Regulation of UCP2 is AMPK- and PPARγ-dependent in MTO1 fibroblasts. (A) qRT-PCR analysis of the UCP2 mRNA expression in MTO1 human fibroblasts (MTO1 HF), treated or not with 5 μM rosiglitazone (RGZ) for 1 h. Data are represented as fold change respect to WT HF values. (B) ). The scatter plot shows densitometric data for UCP2 normalized to porin and phosphor-Thr172-AMPKα normalized to AMPKα, and represented as fold change relative to WT HF. (C) ). The scatter plot shows densitometric data for UCP2 normalized to porin and phosphor-Thr172-AMPKα normalized to AMPKα, and represented as fold change relative to WT HF. All data are the mean ± SD of at least three different experiments. Differences from WT HF values were found to be statistically significant at *p

    Article Snippet: For mRNA quantification, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Quantitative RT-PCR, Expressing

    Angiogenin sensitivity of mt-tRNAs purified from MTO1 defective cells. (A and B) Northern analysis of mt-tRNA Lys (upper panel) and mt-tRNA Val (lower panel) molecules after in vitro angiogenin (ANG) digestion of small RNAs purified from wild-type (WT HF) and MTO1 (MTO1 HF) human fibroblasts ( A ), and from MTO1 siRNA 1- and Negative Control (NC) siRNA-transfected 143B cells ( B ) for 1, 2 and 3 h. Full-length blots are included in supplementary information (Fig. S17). The kinetic analysis of angiogenin digestions are plotted below the representative northern blots. The amount of intact mt-tRNA after 0, 1, 2 and 3 h of incubation with angiogenin is represented as fold change relative to the undigested control (0 h). ( C) qRT-PCR analysis of MTO1 mRNA in MTO1 HF. Data are expressed as fold change respect to WT HF. (D) Representative immunoblot of MTO1 protein expression in MTO1 HF and WT HF. The membrane was also probed with an antibody against porin, which was used as a loading control. Full-length western blots are included in supplementary information (Fig. S18). The scatter plot shows the densitometric analysis of MTO1 normalized to the loading control and represented as fold change respect to WT HF. All data are the mean ± SD of at least three independent biological replicates. Differences from WT or NC values were found to be statistically significant at *p

    Journal: Scientific Reports

    Article Title: Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis

    doi: 10.1038/s41598-018-19587-5

    Figure Lengend Snippet: Angiogenin sensitivity of mt-tRNAs purified from MTO1 defective cells. (A and B) Northern analysis of mt-tRNA Lys (upper panel) and mt-tRNA Val (lower panel) molecules after in vitro angiogenin (ANG) digestion of small RNAs purified from wild-type (WT HF) and MTO1 (MTO1 HF) human fibroblasts ( A ), and from MTO1 siRNA 1- and Negative Control (NC) siRNA-transfected 143B cells ( B ) for 1, 2 and 3 h. Full-length blots are included in supplementary information (Fig. S17). The kinetic analysis of angiogenin digestions are plotted below the representative northern blots. The amount of intact mt-tRNA after 0, 1, 2 and 3 h of incubation with angiogenin is represented as fold change relative to the undigested control (0 h). ( C) qRT-PCR analysis of MTO1 mRNA in MTO1 HF. Data are expressed as fold change respect to WT HF. (D) Representative immunoblot of MTO1 protein expression in MTO1 HF and WT HF. The membrane was also probed with an antibody against porin, which was used as a loading control. Full-length western blots are included in supplementary information (Fig. S18). The scatter plot shows the densitometric analysis of MTO1 normalized to the loading control and represented as fold change respect to WT HF. All data are the mean ± SD of at least three independent biological replicates. Differences from WT or NC values were found to be statistically significant at *p

    Article Snippet: For mRNA quantification, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Purification, Northern Blot, In Vitro, Negative Control, Transfection, Incubation, Quantitative RT-PCR, Expressing, Western Blot

    MTO1 and GTPBP3 defects produce opposite effects on HIF-1 signaling. ( A,D and G) qRT-PCR analysis of the HIF-1 mRNA expression in MTO1 HF ( A ), in MTO1 siRNA1- and MTO1 siRNA 2-transfected 143B cells ( D ), and in GTPBP3 siRNA1- and GTPBP3 siRNA 2-transfected 143B cells ( G ). (B,E and H) Representative immunoblots of HIF-1 in MTO1 HF ( B ), in MTO1 siRNA1- and MTO1 siRNA 2-transfected cells ( E ), and in GTPBP3 siRNA1- and GTPBP3 siRNA 2-transfected cells ( H ). The scatter plot shows densitometric data for HIF-1 normalized to porin, and represented as fold change relative to WT HF ( B ) or NC siRNA-transfected cells ( E and H ). (C , F and I) qRT-PCR analysis of mRNA expression of the HIF-1 target genes vascular endothelial growth factor ( VEGF ) and platelet derived growth factor subunit 2 ( PDGF2 ) in MTO1 HF ( C ), in MTO1 siRNA1- and MTO1 siRNA 2-transfected cells ( F ), and in GTPBP3 siRNA1- and GTPBP3 siRNA 2-transfected cells ( I ). All data are the mean ± SD of at least three different experiments and are represented as fold change respect to WT HF or NC siRNA-transfected cells. Differences from WT or NC values were found to be statistically significant at *p

    Journal: Scientific Reports

    Article Title: Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis

    doi: 10.1038/s41598-018-19587-5

    Figure Lengend Snippet: MTO1 and GTPBP3 defects produce opposite effects on HIF-1 signaling. ( A,D and G) qRT-PCR analysis of the HIF-1 mRNA expression in MTO1 HF ( A ), in MTO1 siRNA1- and MTO1 siRNA 2-transfected 143B cells ( D ), and in GTPBP3 siRNA1- and GTPBP3 siRNA 2-transfected 143B cells ( G ). (B,E and H) Representative immunoblots of HIF-1 in MTO1 HF ( B ), in MTO1 siRNA1- and MTO1 siRNA 2-transfected cells ( E ), and in GTPBP3 siRNA1- and GTPBP3 siRNA 2-transfected cells ( H ). The scatter plot shows densitometric data for HIF-1 normalized to porin, and represented as fold change relative to WT HF ( B ) or NC siRNA-transfected cells ( E and H ). (C , F and I) qRT-PCR analysis of mRNA expression of the HIF-1 target genes vascular endothelial growth factor ( VEGF ) and platelet derived growth factor subunit 2 ( PDGF2 ) in MTO1 HF ( C ), in MTO1 siRNA1- and MTO1 siRNA 2-transfected cells ( F ), and in GTPBP3 siRNA1- and GTPBP3 siRNA 2-transfected cells ( I ). All data are the mean ± SD of at least three different experiments and are represented as fold change respect to WT HF or NC siRNA-transfected cells. Differences from WT or NC values were found to be statistically significant at *p

    Article Snippet: For mRNA quantification, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Derivative Assay