Fig 2: Oxidized ATM-mediated EMR fuels Ac-CoA production.a, b Western blotting show the glycolysis-related (a) and TCA-cycle-related (b) gene expressions in mammospheres derived from Hs578T and BT549 cells under treatment with or without KU60019 (10 µM) and from ATM-silenced cells in hypoxia. c–h Acetyl-CoA productions of the indicated mammospheres were determined. c Knockdown of GLUT1, PKM2, or PDHa and the control mammospheres. d Ectopic GLUT1, PKM2, or PDHa was transfected into hypoxic mammospheres with ATM knocked down. e Mammospheres treated with or without 2-deoxyglucose (2-DG, 5 mM), UK5099 (an inhibitor of the mitochondrial pyruvate carrier (MPC), 10 µM), or BMS303141 (an inhibitor of ACLY, 20 µM); f mammospheres treated with or without Etomoxir (ETO, an inhibitor of CPT1, 20 µM); g exogenous addition with or without acetate (1, 5, or 10 mM). h Exogenous addition with or without acetate (10 mM) in mammospheres under ATM, GLUT1, PKM2, or PDHa knocked down. i, j A positive correlation between p-ATM and SOX2 protein levels (i), p-ATM and production of acetyl-CoA (j), and SOX2 protein expression and acetyl-CoA (k) in 92 clinical breast cancer samples (*P < 0.05; **P < 0.01).

Fig 3: miR-149-3p/PDK2 signaling inhibits glucose metabolism and increases chemosensitivity. a PDK2 was knocked down by transient transfection with an siRNA in HCT-8/F cells. The knockdown resulted in the alteration of p-PDHA1. b The glucose consumption, lactate production and glycolysis of CRC cells transfected with PDK2 siRNA or NC were measured. c HCT-8/F cells were treated with or without 50 µg/ml 5-FU for 24 h after transfection with PDK2 siRNA or NC. The recognized biomarkers (c-PARP, Bax) for cell apoptosis were determined by Western blot analysis. d HCT-8/F cells transfected with PDK2 siRNA or NC were treated with 5-FU. The cell growth was determined by a CCK8 assay. e HCT-8/F cells infected with PDK2 shRNA or the negative control virus were treated with 5-FU. The colony formation capacity was determined by crystal violet staining. f HCT-8/F cells were transfected with NC or the miR-149-3p mimic for 24 h and then infected with a virus overexpressing PDK2. The expression of PDK2 and p-PDHA1 was determined by Western blot analysis. g Glucose consumption, lactate production and glycolysis were measured. h, i After infection, cells were treated with 5-FU and the cell growth and capacities of colony formation were measured. Representative results of three independent experiments performed in at least triplicate are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 4: Oxidized ATM-mediated EMR promotes tumor formation of CSCs in mice xenografts.a Photos of the xenograft tumors dissected from the nude mice at the seventh week after being subcutaneously injected with BT549 mammosphere cells. When tumor volume reaches around 50 mm3, mice were randomized into five groups and administered with KU60019 (50 mg/kg, three times a week), or UK5099 (10 µg/kg, once every 2 days), acetate (150 mg/kg, three times a week), and acetate (250 mg/kg, three times a week) alone or in combination, via intraperitoneal (i.p.) injection. b Tumor growth curve (*P < 0.05, **P < 0.01). c The average tumor weight of each group. d Acetyl-CoA productions of xenograft tumors. e GLUT1, PKM2, PDHa, H4ac, and SOX2 levels were determined in tumor tissues by IHC analyses.

Fig 5: PDK2 is a direct target of miR-149-3p. a Schematic diagram of the protocol used to search for candidate target genes which are predictively regulated by miR-149-3p. b HCT-8 and HCT116 cells were transiently transfected with an miR-149-3p mimic or inhibitor. The mRNA levels of PDK2 and HK1 were analyzed by quantitative real-time PCR. c Diagram of the miR-149-3p putative binding sites in the 3'-UTR of PDK2. The mutant sequences used in the luciferase reporters are indicated in red. d Human embryonic kidney 293 T cells and HCT116 cells were cotransfected with luciferase reporter constructs and the miR-149-3p mimic (50 nmol/L) or miRNA control for 48 h. Relative luciferase activity data were normalized to corresponding control. e HCT-8 and HCT116 cells were transfected with either the miR-149-3p mimic or inhibitor for 48 h, and the expression of PDK2 was determined by Western blot analysis. f HCT116 cells transfected with the miR-149-3p inhibitor or anti-NC were treated with or without DCA for 24 h. The expression of PDK2 was determined by Western blot analysis. g The expression of PDK2 and p-PDHA1 in chemosensitive and chemoresistant CRC cells were analyzed by Western analysis. Data shown are representative pictures of three experiments. The results of three independent experiments performed in triplicate are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001