Wang JJ , Wang Y , Hou L , Xin F , Fan B , Lu C , Zhang L , Wang F , Li S .

5182027 Beijing Municipal Natural Science Foundation

	Figure 2. Typical 1H NMR spectra (δ0.5-9.0) of A549 cell extracts obtained from groups treated with (S) and without (C) rFIP-nha. The region of δ6.0–9.5 (in the dashed box) was magnified 20 times compared with the corresponding region of δ0.5–6.0 for clarity. Keys: 1-MH, 1-methylhistideine; 3-HB, 3-hydroxybutyrate; AA, acetoacetate; Ace, acetate; Act, acetone; Ala, alanine; Bu, butyrate; Cho, choline; Cit, citrate; Cn, creatinine; Cr, creatine; EA, ethanolamine; Eth, ethanol; For, formate; G, glycerol; Glc, glucose; Gln, glutamine; His, histidine; HIV, 3-hydroxyisovalerate; HOD, the residual water signals; IB, isobutyrate; Ile, isoleucine; KIV, 2-ketoisovalerate; Lac, lactate; Lys, lysine; Mal, mannitol; MG, 7-methylguanosine; MNA, 1-methylnicotinamide; Mol, methanol; p-AB, p-aminobenzoate; PC, phosphocholine: Phe, phenylalanine; Py, pyruvate; SCFA, short-chain fatty acid; Tau, Taurine; Thr, threonine; TMA, trimethylamine; TMAO, trimethylamine N-oxide; Tyr, tyrosine; Val, valine.
	Figure 3. (A) 2D principal component analysis (PCA) scores plots based on 1H NMR spectra of A549 cells obtained from groups C and S. (B) Partial least squares-discriminant analysis (PLS-DA) scores plots based on 1H NMR spectra of A549 cells obtained from groups C and S. The experimental group of A549 cells (group S) were treated with 16 μg/mL FIP-nha for 24 h, and the control group (group C) was treated with same volume of PBS for 24 h.
	Figure 4. Orthogonal projection to latent structure with discriminant analysis (OPLS-DA) scores plots (A) derived from 1H NMR spectra of A549 cells. Corresponding coefficient loading plots (B) obtained from groups C and S and cross validation (C) by permutation test (n = 200). The significance of metabolites variations between the two classes is shown in a color map. Peaks in the negative direction demonstrate metabolites of group C are more abundant. In the group S, if metabolites are more abundant, the peaks will in the positive direction. Figure 2 shows the keys of the assignment.
	Figure 5. Summary of altered metabolic pathways and regulatory mechanism of rFIP-nha against A549 cells. HK, hexokinase; PGI, phosphoglucose isomerase; PFK1, phosphofructokinase 1; Aldo, aldolase; TPI, triose phosphate isomerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; Eno, enolase; PK, pyruvate kinase; LDH, lactate dehydrogenase; TCA, tricarboxylic acid cycle; G6PD, glucose-6-phosphate dehydrogenase; TPA, taurine-pyruvate aminotransferase; pfp, pyrophosphate-fructose-6-phosphate 1-phosphotransferase; mtlD, mannitol-1-phosphate 5-dehydrogenase; M1Pase, mannitol-1-phosphatase; gldA, glycerol dehydrogenase; DAK, dihydroxyacetone kinase; GLUTs, glucose transporters; HER2, receptor tyrosine-protein kinase erbB-2; PI3K, phosphatidylinositol 3-kinase; Akt, serine-threonine kinase; mTOR, mammalian target of rapamycin; HIF-1α, hypoxia-inducible factor 1α; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; c-Myc, a transcription factor; p53, a tumor suppressor; Pten, a human tumor suppressor gene on chromosome 10; AMPK, AMP-activated protein kinase; TSC2, tuberous sclerosis complex 2; TIGAR, TP53-induced glycolysis and apoptosis regulator; GLS2, glutaminase 2; SCO2, cytochrome c oxidase 2; COX4, cytochrome c oxidase subunit 4. HER2-mediated PI3K-Akt-mTOR signaling plays a pivotal role in promoting glycolysis in tumor cells via the activation of HIF-1α, NF-κB, and c-Myc. p53 plays a key role in the process of suppressing glycolysis and promoting oxidative phosphorylation (OXPHOS) by interacting with various enzymes and other molecules, including SCO2, TIGAR, GLUT1, GLUT4, GLS2, and PGM. Different metabolic pathways represented within the dashed box. The arrow represents stimulatory modification, the T-shaped arrow represents inhibitory modification.

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