Knocking out MIF exacerbates Cfz-induced mitochondrial swelling and superoxide production in MM cells. Confocal immunofluorescent imaging (A) and summarized data (B) showing mitochondrial morphology of CTR-KO or MIF-KO MM.1S or ARP-1 MM cells using MitoTracker Red CMXRos (red) and 4′,6-diamidino-2-phenylindole (blue) staining after DMSO or Cfz treatment of 16 hours. One representative of at least 4 fields is shown. (C) Pie chart showing the percentage of oxidative stress–related metabolites (labeled as red) among the top 22 upregulated metabolites in supplemental Figure 3C. (D) Heatmap showing the fold change (FC) of oxidative stress–related metabolites in Cfz-treated MIF-KO vs CTR-KO ARP-1 MM cells. Flow cytometry histogram (E) and summarized data showing MitoSOX Red fluorescence in CTR-KO or MIF-KO MM.1S (F), ARP-1 (G), or KMS-11/Cfz (H) MM cells after pulse treatment with DMSO or Cfz for 16 hours. CTR-KO or MIF-KO MM.1S or ARP-1 MM cells were pulsed with DMSO or Cfz for 1 hour and cultured in Cfz-free medium without or with N-acetylcysteine (NAC) (N + C) for 24 hours; the apoptotic rates are shown in a flow cytometry histogram (I) and as summarized results (J-K). For panels E and I, one representative result of at least 3 independent experiments is shown. The Student t test was used to compare 2 samples. *P < .05; **P < .01. n.s., not significant.