Figure 4.
Immunometabolic changes indicative of TI in macrophages expressing BRAFV600E. (A) Western blot determination of phosphorylated AKT (p-AKT). The levels of total AKT in whole-cell lysates were used as the protein-loading control. (B) Analysis of ECAR measurements on primary human monocytes isolated from healthy volunteers in 3 different conditions, nontransduced (UT), transduced with wtBRAF (WT) or BRAFV600E (VE), in basal conditions and after sequential addition of glucose, oligomycin, and 2-DG. (C) Relative ECAR to oxygen consumption rate ratio (right). ***P < .001; ****P < .0001, unpaired Student t test. (D) Summary of metabolomics studies: primary human monocytes isolated from 3 healthy volunteers were cultured in 3 different conditions: nontransduced (UT, isolated primary monocytes), transduced with wtBRAF (WT), and transduced with BRAFV600E (VE). Cells were cultured in RPMI-1640+FBS only (Cold) or RPMI-1640+FBS with the addition of 13C6-glucose labeled glucose or 13C5-glutamine for tracing experiments. (E) Principal component analysis (PCA) of high-throughput metabolomics analysis on cold samples showing separation of experimental conditions along the component 1 axis (76.3%); interindividual variability is described by the component 2 axis (12.8%). (F) Hierarchical clustering analysis of the top 50 significant metabolites (Student t test) is represented as a heat map grouped by pathways that are known to be relevant to TI. (G) Overview of the glycolysis pathway in tracing experiments with U-13C6-glucose. (H) Overview of the TCA pathway in tracing experiments with 13C5-glutamine. (I) Incorporation of carbons in de novo synthesized cholesterol: carbon molecules deriving from either labeled glucose (red) or labeled glutamine (blue) are expressed as a percentage of the total. (J) Hematoxylin and eosin stain of ECD lesion (skin biopsy specimen, original magnification x200). (G-I) *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, nonsignificant. Statistical significance of differences was evaluated with ANOVA. FBS, fetal bovine serum.

Immunometabolic changes indicative of TI in macrophages expressing BRAFV600E. (A) Western blot determination of phosphorylated AKT (p-AKT). The levels of total AKT in whole-cell lysates were used as the protein-loading control. (B) Analysis of ECAR measurements on primary human monocytes isolated from healthy volunteers in 3 different conditions, nontransduced (UT), transduced with wtBRAF (WT) or BRAFV600E (VE), in basal conditions and after sequential addition of glucose, oligomycin, and 2-DG. (C) Relative ECAR to oxygen consumption rate ratio (right). ***P < .001; ****P < .0001, unpaired Student t test. (D) Summary of metabolomics studies: primary human monocytes isolated from 3 healthy volunteers were cultured in 3 different conditions: nontransduced (UT, isolated primary monocytes), transduced with wtBRAF (WT), and transduced with BRAFV600E (VE). Cells were cultured in RPMI-1640+FBS only (Cold) or RPMI-1640+FBS with the addition of 13C6-glucose labeled glucose or 13C5-glutamine for tracing experiments. (E) Principal component analysis (PCA) of high-throughput metabolomics analysis on cold samples showing separation of experimental conditions along the component 1 axis (76.3%); interindividual variability is described by the component 2 axis (12.8%). (F) Hierarchical clustering analysis of the top 50 significant metabolites (Student t test) is represented as a heat map grouped by pathways that are known to be relevant to TI. (G) Overview of the glycolysis pathway in tracing experiments with U-13C6-glucose. (H) Overview of the TCA pathway in tracing experiments with 13C5-glutamine. (I) Incorporation of carbons in de novo synthesized cholesterol: carbon molecules deriving from either labeled glucose (red) or labeled glutamine (blue) are expressed as a percentage of the total. (J) Hematoxylin and eosin stain of ECD lesion (skin biopsy specimen, original magnification x200). (G-I) *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, nonsignificant. Statistical significance of differences was evaluated with ANOVA. FBS, fetal bovine serum.

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