Fig 2: MLH1 loss is associated with reduced mitochondrial gene expression.Quantitative RT-PCR analysis of RNA extracted from a HCT116 and HCT116+ chr3 cells or b KLE cells transfected with either siCTRL siRNA targeting MLH1. mRNA expression was measured using PGC1ß and ß-actin Taqman probes. ß-actin was used as a control. Quantitative RT-PCR analysis of RNA extracted from c HCT116 and HCT116+ chr3 cells or d KLE cells transfected with either siCTRL or siRNA targeting MLH1. mRNA expression was measured using MLH1, NRF2, GPX1 and ß-actin Taqman probes. ß-actin was used as a control. e Western blot analysis of MLH1-deficient HCT116 and MLH1-proficient HCT116+ chr3 cells. Protein was extracted and expression was analysed using antibodies as indicated. ß-actin is used as a loading control. f GPX1 activity was measured using a commercially available GPX1 activity assay. Protein lysates were isolated from the KLE cells transfected with either siControl or siRNA targeting MLH1. Equal amounts of protein were incubated to determine the activity of GPX1 by measuring the optical density for NADPH at 340 nm before and after addition of cumene hydroperoxide (substrate for GPX1)

Fig 3: Reducing equivalents flow from glucose to Se-dependent antioxidant enzymes, GPx1 and GPx4. In cells and tissue, the vast majority of GPx1 and GPx4 exists in a fully reduced state, poised to transfer electrons to hydroperoxides, thereby removing them and eliminating their potential to initiate new chains of detrimental oxidations. Both enzymes are reduced back to the active form by 2GSH. Using electrons from NADPH, the GSSG formed is reduced back to 2GSH by glutathione disulfide reductase (GR). Through the pentose phosphate pathway, NADP+ is reduced back to NADPH. Thus, oxidized GPxs are “recycled”.

Fig 4: GPx1 and GPx4 protein expression and activities are controlled by the amount of FBS in cell culture media. (A) GPx4 protein expression in MIA PaCa-2 cells is decreased when grown in lower concentrations of FBS (1–10%). But can be restored and optimized by adding 200 nM SLM for 48 h. (B) GPx1 and GPx4 activities are decreased with FBS concentrations < 10% in MIA PaCa-2 cells. Supplementation with SLM increases both GPx1 and GPx4 activities, each to the same level no matter the percent of FBS used. (Note the different scales for the ordinates.) (C) GPx4 protein expression in HepG2 cells is decreased when grown in lower concentrations of FBS (1–10%). But can be restored and optimized by adding 200 nM SLM for 48 h. (D) GPx1 and GPx4 activities are decreased with FBS concentrations < 10% in HepG2 cells. Supplementation with SLM increases both GPx1 and GPx4 activities, each to the same level no matter the percent of FBS used. (Note the different scales for the ordinates.).

Fig 5: Protein expression levels before and after 4 days bed rest, a representative blot is shown on each figure. Data are means ± SD. *P < 0.05. Black circles represents prebed rest; gray squares represents postbed rest. GPX1 indicates glutathione peroxidase 1; MFN2, mitofusin‐2; SOD, superoxide dismutase.