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. 2023 Feb 16;15(6):1325–1350. doi: 10.1016/j.jcmgh.2023.02.004

Figure 6.

Figure 6

PFKFB4 functions as a phosphatase in HCC cells and its loss resulted in the accumulation of glycolytic metabolites and distorted PPP. (A) Principal component analysis illustrating the overall differences between the metabolomic profiles between the PFKFB4 KO and control Huh7 cells under normoxic and hypoxic conditions. (B) Unsupervised hierarchical clustering analysis of the metabolomic profiles between the PFKFB4 KO and control Huh7 cells under normoxic and hypoxic conditions. (C) Schematic diagram showing the key metabolic enzymes in glycolysis and PPP and their metabolic substrates and products. (D) The concentration of glycolytic intermediates in PFKFB4 KO and control Huh7 cells under normoxic and hypoxic conditions. (E) PFKFB4 KO and control cells were cultured with glucose-free medium and subjected to hypoxic treatment for 24 hours, followed by incubation with 2-NDBG, the fluorescently labeled glucose analog, for 10 minutes, followed by flow cytometry. The relative 2-NDBG intensities per cell between the PFKFB4 KO and control cells are shown. (F) The overall glycolytic rate and oxidative phosphorylation rate between PFKFB4 KO and control cells was assessed by the Seahorse glycolytic rate test kit on a Seahorse bioanalyzer. The relative glycolytic rate as a function of the extracellular acidification rate (ECAR) (top panel) and the oxidative phosphorylation rate as a function of the oxygen consumption rate (OCR) (bottom panel) are shown. (G) The concentration of PPP and purine metabolism intermediates in PFKFB4 KO and control Huh7 cells under normoxic and hypoxic conditions. (H) The concentration of NADPH and glutathione in PFKFB4 KO and control Huh7 cells under normoxic and hypoxic conditions. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. DHAP, Dihydroxyacetone phosphate; PC, Principal component; PEP, Phosphoenolpyruvate.