Fig 2: MIF mRNA and protein expression in five lung squamous cell carcinoma cell lines. MIF mRNA and protein were detected in all cell lines with higher expression in HCC‐1588 compared with those of others.

Fig 3: Glycation and oxidation of MIF.(a) Coomasie stained SDS-PAGE (top) and Flu-PAGE (bottom) analysis of recombinant MIF after glycation with glucose (50 mM) at 37 °C for 7 days. The MIF doublet can be observed developing over the time course, and is seen much earlier by Flu-PAGE. (b) Flu-PAGE analysis of recombinant MIF after glycating with methylglyoxal (50 mM) at 37 °C for 7 days. The MIF doublet was not reproducible using methylglyoxal, and can be seen to aggressively induce protein cross-linking. Molecular weight (MW) is given in kDa (c) MIF incubated in PBS alone, or in the presence of GSH (10 mM) or DTT (5 mM) at 37 °C for 4 days. Co-incubation of MIF with an antioxidant prevented or slowed the formation of the MIF doublet. (d) Oxidation of MIF by incubating in the presence of H2O2 (1 mM), GSSG (10 mM) or GSNO (400 μM) at 37 °C for 1 hour. The MIF doublet could be mimicked by H2O2 and GSNO under the conditions described. (e) Trypsin digest of unmodified and glycated MIF with glucose or MG, followed over 4 hours at 37 °C. Under these conditions, glycation was able to reduce or inhibit proteolysis by trypsin. Full-length gel images for Fig. 2(a), (c), (d) and (e) are included in Supplementary Figures S7–S9.

Fig 4: Wound healing assay on MIF status in HCC‐1588 cells. The cells with knockdown of MIF showed much slower migration into the wound area than control cells. Data represent at least two independent experiments with similar results. KD, knockdown.

Fig 5: MIF enzyme activity affected by glycation and oxidation.(a) Tautomerase activity of MIF measured by the tautomerisation of L-dopachrome methyl ester at 450 nm. Data represents activity relative to unmodified MIF. Oxidation and glycation of MIF are observed to have a significant negative effect on tautomerase activity, with methylglyoxal (MG) completely inhibiting the catalysis. Errors are represented as s.e.m. with n = 7 or 8. Student’s t-test ****P = < 0.0001, **P = 0.0012. (b) Insulin reduction assay, following the MIF catalysed precipitation of the insulin b chain at 650 nm. Glycated MIF was unable to catalyse the reduction of insulin by GSH, while (c) in vitro oxidised MIF showed no significant (ns) difference in activity relative to unmodified MIF. Plot shows the reaction endpoint, with s.e.m. and n = 11. Student’s t-test **P = 0.0036. (d) Ellman’s test to determine the free thiol content of MIF under native and denaturing conditions. Oxidation under these conditions did not affect the free thiol content, and glycation showed a small reduction in available thiols. Errors expressed as s.e.m., n = 3. Student’s t-test ***P < 0.0003. Glycated MIF was produced by incubation at 37 °C with glucose, oxidised MIF was produced by incubation at 37 °C without glucose, as described in text. Raw tautomerase activity and insulin reduction assays are shown in Supplementary Figure S5.