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    Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using <t>tcaCALC.</t> All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.
    Tcacalc Program, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Regulation of Metabolism by Mitochondrial MUL1 E3 Ubiquitin Ligase"

    Article Title: Regulation of Metabolism by Mitochondrial MUL1 E3 Ubiquitin Ligase

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.904728

    Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.
    Figure Legend Snippet: Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.

    Techniques Used: Labeling, Derivative Assay

    Akt2 and HIF-1α proteins contribute to support the metabolic phenotype of HEK293 MUL1(−/−) cells. (A) Glycolytic capacity in HEK293 WT and Akt2(−/−) cells before or after treatment with the HIF-1α activator DMOG (100 μM for 4 h). Extracellular acidification rate (ECAR) was measured using the Seahorse analyzer. HEK293 MUL1(−/−) cells were used as a positive control. #, p ≤ 0.035: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (B) Quantification of the glycolysis, glycolytic capacity, and glycolytic reserve obtained from three independent experiments. Data from three separate experiments are presented as means ± SEM. #, p ≤ 0.03: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (C) The effect of HIF-1α activator, DMOG, on the expression levels of HIF-1α, GLUT1, Akt2, P-GSK-3β S9, in Akt2(−/−), or MUL1(−/−) cells was monitored by western blot analysis. (D) Densitometrical analysis of the proteins shown in panel (C), normalized against β-actin. Results shown as means ± SD of three independent experiments. *, p ≤ 0.03 vs . WT con; #, p ≤ 0.025 vs . Akt2(−/−) con. (E) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of Akt2(−/−) and Akt2(−/−) +DMOG cells utilizing the [U- 13 C]glucose (note: S, D, T, and Q are singlet, doublet, triplet, and quartet, respectively. All signal ratios were calculated with respect to the total area of the corresponding glutamate resonance. Data are represented as mean ± SEM. (F) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, for Akt2(−/−) vs. Akt2(−/−)+DMOG.
    Figure Legend Snippet: Akt2 and HIF-1α proteins contribute to support the metabolic phenotype of HEK293 MUL1(−/−) cells. (A) Glycolytic capacity in HEK293 WT and Akt2(−/−) cells before or after treatment with the HIF-1α activator DMOG (100 μM for 4 h). Extracellular acidification rate (ECAR) was measured using the Seahorse analyzer. HEK293 MUL1(−/−) cells were used as a positive control. #, p ≤ 0.035: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (B) Quantification of the glycolysis, glycolytic capacity, and glycolytic reserve obtained from three independent experiments. Data from three separate experiments are presented as means ± SEM. #, p ≤ 0.03: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (C) The effect of HIF-1α activator, DMOG, on the expression levels of HIF-1α, GLUT1, Akt2, P-GSK-3β S9, in Akt2(−/−), or MUL1(−/−) cells was monitored by western blot analysis. (D) Densitometrical analysis of the proteins shown in panel (C), normalized against β-actin. Results shown as means ± SD of three independent experiments. *, p ≤ 0.03 vs . WT con; #, p ≤ 0.025 vs . Akt2(−/−) con. (E) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of Akt2(−/−) and Akt2(−/−) +DMOG cells utilizing the [U- 13 C]glucose (note: S, D, T, and Q are singlet, doublet, triplet, and quartet, respectively. All signal ratios were calculated with respect to the total area of the corresponding glutamate resonance. Data are represented as mean ± SEM. (F) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, for Akt2(−/−) vs. Akt2(−/−)+DMOG.

    Techniques Used: Positive Control, Expressing, Western Blot



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    tcacalc program  (MathWorks Inc)
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    MathWorks Inc tcacalc program
    Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using <t>tcaCALC.</t> All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.
    Tcacalc Program, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tcacalc program/product/MathWorks Inc
    Average 90 stars, based on 1 article reviews
    tcacalc program - by Bioz Stars, 2026-05
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    Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Regulation of Metabolism by Mitochondrial MUL1 E3 Ubiquitin Ligase

    doi: 10.3389/fcell.2022.904728

    Figure Lengend Snippet: Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.

    Article Snippet: The tcaCALC program in MATLAB was utilized to perform an isotopomer analysis to estimate relative pathway fluxes ( ).

    Techniques: Labeling, Derivative Assay

    Akt2 and HIF-1α proteins contribute to support the metabolic phenotype of HEK293 MUL1(−/−) cells. (A) Glycolytic capacity in HEK293 WT and Akt2(−/−) cells before or after treatment with the HIF-1α activator DMOG (100 μM for 4 h). Extracellular acidification rate (ECAR) was measured using the Seahorse analyzer. HEK293 MUL1(−/−) cells were used as a positive control. #, p ≤ 0.035: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (B) Quantification of the glycolysis, glycolytic capacity, and glycolytic reserve obtained from three independent experiments. Data from three separate experiments are presented as means ± SEM. #, p ≤ 0.03: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (C) The effect of HIF-1α activator, DMOG, on the expression levels of HIF-1α, GLUT1, Akt2, P-GSK-3β S9, in Akt2(−/−), or MUL1(−/−) cells was monitored by western blot analysis. (D) Densitometrical analysis of the proteins shown in panel (C), normalized against β-actin. Results shown as means ± SD of three independent experiments. *, p ≤ 0.03 vs . WT con; #, p ≤ 0.025 vs . Akt2(−/−) con. (E) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of Akt2(−/−) and Akt2(−/−) +DMOG cells utilizing the [U- 13 C]glucose (note: S, D, T, and Q are singlet, doublet, triplet, and quartet, respectively. All signal ratios were calculated with respect to the total area of the corresponding glutamate resonance. Data are represented as mean ± SEM. (F) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, for Akt2(−/−) vs. Akt2(−/−)+DMOG.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Regulation of Metabolism by Mitochondrial MUL1 E3 Ubiquitin Ligase

    doi: 10.3389/fcell.2022.904728

    Figure Lengend Snippet: Akt2 and HIF-1α proteins contribute to support the metabolic phenotype of HEK293 MUL1(−/−) cells. (A) Glycolytic capacity in HEK293 WT and Akt2(−/−) cells before or after treatment with the HIF-1α activator DMOG (100 μM for 4 h). Extracellular acidification rate (ECAR) was measured using the Seahorse analyzer. HEK293 MUL1(−/−) cells were used as a positive control. #, p ≤ 0.035: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (B) Quantification of the glycolysis, glycolytic capacity, and glycolytic reserve obtained from three independent experiments. Data from three separate experiments are presented as means ± SEM. #, p ≤ 0.03: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (C) The effect of HIF-1α activator, DMOG, on the expression levels of HIF-1α, GLUT1, Akt2, P-GSK-3β S9, in Akt2(−/−), or MUL1(−/−) cells was monitored by western blot analysis. (D) Densitometrical analysis of the proteins shown in panel (C), normalized against β-actin. Results shown as means ± SD of three independent experiments. *, p ≤ 0.03 vs . WT con; #, p ≤ 0.025 vs . Akt2(−/−) con. (E) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of Akt2(−/−) and Akt2(−/−) +DMOG cells utilizing the [U- 13 C]glucose (note: S, D, T, and Q are singlet, doublet, triplet, and quartet, respectively. All signal ratios were calculated with respect to the total area of the corresponding glutamate resonance. Data are represented as mean ± SEM. (F) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, for Akt2(−/−) vs. Akt2(−/−)+DMOG.

    Article Snippet: The tcaCALC program in MATLAB was utilized to perform an isotopomer analysis to estimate relative pathway fluxes ( ).

    Techniques: Positive Control, Expressing, Western Blot