Role of MIF homotrimers in SOD1 folding and activity. MIF-KO ARP-1 MM cells were induced to re-express WT or indicated mutant MIFs or empty vectors (Vec) and treated with Cfz. Cfz-treated MIF-KO ARP-1 MM cells without lentivirus infection served as mock and CTR-KO ARP-1 MM cells treated with Cfz served as a control. (A) Western blot showing the expression of misfolded SOD1, detected by immunoblotting of immunoprecipitates using the B8H10 monoclonal antibody, and total SOD1, MIF, and β-actin. (B) Quantification of relative band density of misfolded SOD1/input SOD1 (n = 3). (C) SOD1 activity detected by using enzyme-linked immunosorbent assay. Cell apoptosis shown as flow cytometry histogram (D) or summarized results (E). ARP-1 MM cells were pulsed with DMSO or Cfz for 1 hour and then treated without or with ebselen (Ebs + Cfz). Misfolded SOD1 expression (F) and SOD1 activity (G) were examined at 16 hours’ posttreatment; the percentage of superoxide⁺Annexin V⁺ population in ARP-1 MM cells at 24 hours after treatment are shown by dot plot (H) and summarized results (I). (J) Summarized data showing superoxide⁺Annexin V⁺ population in primary MM cells (n = 6) treated with DMSO, 10 μM ebselen, Cfz, or Ebs+Cfz, normalized with DMSO-treated group. For panels D and H, 1 representative result of at least 3 independent experiments is shown. The Student t test was used to compare 2 samples. *P < .05; **P < .01. a.u., arbitrary units; LT, long exposure time; n.s., not significant; ST, short exposure time.