Figure 4.

MIF regulates SOD1 folding and activity important for MM response to Cfz. (A) Gene set enrichment analysis of the top 6 representative Hallmark gene sets (with indicated colors) based on the normalized enrichment score (NES) from RNA-sequencing data of Cfz-treated vs DMSO-treated ARP-1 MM cells. (B) Heatmap illustrating the log2-fold change of the top 11 enrichment core genes involved in reactive oxygen species (ROS) pathways. Ratio in messenger RNA (mRNA) expression of SOD1 in MM.1S or ARP-1 MM cells (C), and in CTR-KO or MIF-KO ARP-1 MM cells (D) after DMSO or Cfz treatment of 16 hours. The housekeeping gene GAPDH was used for normalization of the quantitative reverse transcription polymerase chain reaction results. (E) Western blot showing SOD1 expression in CTR-KO or MIF-KO ARP-1 MM cells after DMSO or Cfz treatment of 16 hours. Representative results from 1 of 2 repeated experiments are shown. CTR-KO or MIF-KO MM.1S or ARP-1 MM cells were pulsed with DMSO or Cfz for 1 hour. After 16 hours, misfolded SOD1 (F) was detected by immunoblotting of immunoprecipitates with anti-misfolded SOD1 B8H10 monoclonal antibody, and SOD1 activity (G) was determined by using enzyme-linked immunosorbent assay. Histogram (H) and summarized data (I) showing apoptosis in MM.1S, ARP-1, and KMS-11/Cfz MM cells treated with DMSO, 0.5 μM ATN-224, Cfz, or ATN-224 plus Cfz (ATN+Cfz) for 24 hours. For panel 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. FDR, false discovery rate; n.s., not significant.