Figure 1. The polyol and pentose phosphate pathways increase the concentration of intracellular reactive oxygen species (ROS) in ENO1-silenced cells.

A. Heat map of proteins differentially expressed in shENO1 compared to shCTRL CFPAC-1 cells (Genesis software). Based on the spectra count label-free quantitation approach, LC-MS/MS analysis identified 13 up-regulated (red) and 8 down-regulated (green) proteins involved in metabolism. Other identified proteins are shown in Supplementary Figure S2A. B. Schematic flow chart of the first steps of glycolysis and the branched Polyol Pathway (PP) and the Pentose Phosphate Pathway (PPP). Enzymes that were up- or down-modulated by LC-MS/MS analysis in shENO1 cells compared to shCNTRL cells are shown in red (as indicated by arrows). C. Uptake of glucose measured in CFPAC-1, MDA-MB-231 and NCI-H441 cell lines transduced with shCTRL (white bars) or shENO1 (black bars). Uptake was expressed as pmol 2-deoxy-D-[³H]-glucose/mg protein. D. Analysis of lactate levels in shCTRL and shENO1 CFPAC-1, MDA-MB-231 and NCI-H441 cell lines. E.–F. Analysis of aldose reductase (ALDR) activity measured by means of the rate of NADPH oxidation (E), and NADPH oxidase activity assessed by the isoluminol-chemiluminescence assay (F) in shCTRL (white bars) and shENO1 (black bars) cell lines. Chemiluminescence was expressed as relative luminescence unit (RLU)/mg protein. G.–H. Analysis of ROS concentration measured by the DCFDA-AM assay (G) and of [1-¹⁴C] glucose flux through the PPP, assessed by ¹⁴CO₂ release (H) in the presence or absence of inhibitors of the mitochondrial chain (rotenone), NADPH oxidase (apocynin) and ALDR (zopolrestat) in CFPAC-1 (left panels), MDA-MB-231 (middle panels) and NCI-H441 (right panels) cell lines transduced with shCTRL (white bars) or shENO1 (black bars). I. Analysis of NADPH oxidase activity, as described above, after selective inhibition of the PPP by dehydroepiandrosterone (DHEA) in CFPAC-1 (left panels), MDA-MB-231 (middle panels) and NCI-H441 (right panels) cell lines transduced with shCTRL (white bars) or shENO1 (black bars). All the graphs illustrate the mean result of three independent experiments ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 relative to shCTRL.