Fig 2: ROS and Grx1 levels and systems analysis of the differential redox proteome of HepG2PRDX6-/-cell line. A) ROS levels in basal and 100 μM H2O2 treated cells expressed as arbitrary fluorescence units in the DCFDA assay normalized for number of cells (n = 4). B) Volcano plot of the change the in reduced/oxidized ratio of quantified Cys-peptides in PRDX6-knockout HepG2 cells; dotted lines indicate the significance thresholds, q-value<0.05 and fold change ≥1.5 or ≤0.67. C) IPA systems analysis of the redox proteome showing the upstream regulators (filter set as in Fig. 2) predicted to be affected by redox changes in their target proteins, ordered by increasing p-value. D) Grx1 levels determined by Western blot (n = 4). The fold change (FC) determined by the quantitative proteomic analysis (n = 4) is shown on top of the graph; membrane images in which WT (+/+) and KO (−/−) paired samples from different experiments (n = 4) were run in the same gel; membranes were cut at appropriate height according to expected migration of Grx1 (≈12 kDa) to reveal with specific antibody. Loading in each lane was normalized to actin. E) Redox change in Cys79/Cys83 of Grx1 in PRDX6 knockout cells; the reduced/oxidized ratio in shown. Statistical significance was assessed by Student's t-test and is shown with a number of asterisks inversely proportional to the p-value (*** ≤0.001, **≤0.005, *≤0.05).

Fig 3: Mitochondrial functionality of HepG2PRDX6-/-cell line. A) Representative recording of oxygen consumption rate (OCR) during the extracellular flow analysis (“Seahorse”) following the protocol for “Mitochondrial Function”; differences between WT and Prdx6 KO cells are highly significant despite a drop of OCR after addition of FCCP. B) Histogram plots of the calculated basal and maximum respiratory rate, ATP production rate and respiratory capacity reserve, normalized for protein content (n = 4). C) Table with mitochondrial protein components of Complexes I and II that were down-regulated in HepG2PRDX6−/- according to the quantitative proteomic analysis. UniProt ID, Protein name, fold change and statistical score are shown; “-inf” indicates that the protein was detected in normal HepG2 cells but not in PRDX6 knockout cells. D) Extracellular glucose and E) lactate concentrations determined by standard methods normalized for number of cells (n = 4). (**** p-value ≤ 0.0001; ** p-value ≤ 0.005).

Fig 4: Prdx6 inhibits activation of the NF-κB signaling pathway by suppressing the nuclear translocation of P65. A Immunofluorescence analysis of P65 in cells transfected with either Prdx6-siRNA or NC-siRNA. DAPI is shown in blue and represents the nucleus. Scale bar = 100 μm. B Nuclear (N) and cytosolic/membrane (C + M) proteins of the NF-κB pathway in Prdx6-siRNA- or NC-siRNA-transfected cells. The cells in all groups were first pretreated with 40 μg/ml curcumin and DMSO and then changed to 20% OLVafter serum for 48 h. The results are shown as the means ± SD of 3 individual experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 5: Cell cycle state in PRDX6 knockout HepG2 cells. The cell cycle is shown in the middle with a different color for each phase and the phase transitions and checkpoints indicated by intersecting thick short lines; proteins differentially expressed in HepG2PRDX6-/- cells as determined by quantitative proteomics are represented; processes and regulators predicted to be activated or inhibited by systems analysis have also been included; green and red vertical arrows indicate up- and down-regulated proteins, respectively; red crosses indicate inhibition; purple arrows and blunt lines indicate positive or negative action, respectively. Read subsection 3.7. in the main text for a full explanation.