Fig 1: Effects of SIRT1 on ZEA-induced cytotoxicity and lactic acid reduction in SCs. (A) After SCs were treated with RSV and ZEA, cytotoxicity was detected using an LDH release kit. (B) Following incubation with RSV for 24 h, the effects of ZEA on ATP levels were examined using an ATP kit. (C) After SCs were treated with RSV and ZEA for 24 h, the effects of ZEA on intracellular lactate levels were examined by using lactic acid kit. (D) SCs were treated with RSV and ZEA for 24 h, and the effects of ZEA on intracellular pyruvate levels were examined by using a pyruvate kit. (E) Western blot analysis of the expression of related proteins in the lactate production pathway after incubation with RSV for 24 h. (F, G, H) Ratio of GLUT1/GAPDH, LDH/GAPDH, MCT4/GAPDH. *p < 0.05, **p < 0.01 versus the control group. ##p < 0.01 compared to the 20 µM ZEA group.
Fig 2: Distribution and metabolic consequences of L30P/R34P expression in HBs. (A) Expression of NFE2L2, KEAP1, GLUT1, GLUT2, GLUT4, PKM-1, PKM-2, and Cpt1a in 2 representative sets of total lysates from the indicated tissues. (B) Nuclear (N)/cytoplasmic (C) fractionation of the indicated tissues, n = 3–5 samples/group. GAPDH and histone H3 (H3) immunoblots were performed as controls for protein loading and the purity of each fraction. Numbers above the NFE2L2 and KEAP1 panels indicate the fraction of protein associated with each compartment as determined by densitometric scanning of bands. (C) Total OCRs of mitochondria from the indicated tissues in the presence of malate, ADP, pyruvate, glutamate, and succinate. (D) Complex I responses, calculated after addition of rotenone to the reactions in (C) without succinate. (E) Complex II responses as determined from residual activity after addition of rotenone. (F) Responses to pyruvate. (G) Responses to glutamate. (H) ß-FAO responses after addition of malate, L-carnitine, and palmitoyl-CoA. (I) Quantification of mitochondrial DNA (mtDNA) in representative tissues. TaqMan reactions amplified a segment of the mt D-loop region.3,25Each point represents the mean of triplicate TaqMan reactions after normalizing to a control TaqMan reaction for the ApoE nuclear gene. (J) In vitro recovery from oxidative stress. Monolayer cultures of the indicated tumor cells expressing cyto-roGFP or mito-roGFP were exposed to 5 mmol/L hydrogen peroxide (bar) while being monitored by live cell confocal microscope.
Fig 3: HBV infection promotes HBP and increases protein O-GlcNAcylation. (A) Principal component analysis of metabolite profiles obtained using a metabolomics assay in HepG2 cells infected with AdHBV1.3 or AdGFP for 72 h. (B) Heatmap of differentially expressed metabolites subjected to identical treatment conditions as in (A). (C) An overview of the hexosamine biosynthesis pathway (HBP). (D) Relative changes in intermediate metabolites of HBP. (E-F) Relative changes in the levels of UDP-GlcNAc (E) and glucose (F) in HBV-infected HepG2-NTCP cells and HepAD38 cells with tetracycline inducible (Tet-off) HBV expression was determined using the LC-MS/MS targeted metabolomics assay. (G) Immunoblot of total O-GlcNAc from HepG2-NTCP and HepAD38 cells treated for the indicated periods. (H-I) qPCR quantification, n = 3 (H) and immunofluorescence staining (I) of GLUT1 in HepG2-NTCP and HepAD38 cells, DAPI (blue) was used to counterstain nuclei, Scale bar, 10 µm. Data are expressed as the mean ± SD. P values were derived from unpaired, two-tailed Student's t-test in E, F, and H (*P < 0.05,**P < 0.01).
Fig 4: miR-143-5p targets the HIF-1a-associated GLUT1 pathway. (A) miR-143-5p binding site sequences in the 3'-untranslated regions of human HIF-1a detected using TargetScan. (B and C) Overexpression of miR-143-5p decreased the luciferase activity in HIF-1a-WT cells, as measured by the dual-luciferase reporter assay. (D, E and I) miR-143-5p mimics transfection decreased the protein expression levels of GLUT1 and HIF-1a within breast cancer cells, as detected by western blot analysis. (F-H) Pearson's correlation analysis of miR-143-5p, HIF-1a and GLUT1 expression in BT-549 cells. (J-L) Pearson's correlation analysis of miR-143-5p, GLUT1 and HIF-1a expression in MCF-7 cells. Data are expressed as the mean ± SD. *P<0.05. GLUT1, glucose transporter 1; HIF-1a, hypoxia-inducible factor-1a; miR, microRNA; MUT, mutated; NC, negative control; WT, wild type.
Fig 5: Survival analysis of 448 lung adenocarcinoma patients (TCGA-LUAD) and 475 lung squamous cell carcinoma patients (TCGA-LUSC).Median glucose transporter GLUT1 expression value splits down the patient tumors into low and high GLUT1 expressers. Kaplan-Meier plots show that GLUT1 expression is associated with a poor overall survival in patients with (a) LUAD tumors, whereas it is not prognostic in patients with (b) LUSC lesions. p-values were computed with Wald test.
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