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. 2020 Jun 24;136(22):2557–2573. doi: 10.1182/blood.2020005795

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© 2020 by The American Society of Hematology

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Figure 6. — MIF inhibitors sensitize human MM cells to PIs in vitro and in vivo. (A) Cell viability of human MM cell lines pulsed with DMSO or 80 nM Cfz for 1 hour and treated without or with 10 μM 4-IPP (4-IPP+Cfz) for 48 hours, normalized with DMSO-treated group. (B) Summarized results of apoptosis of human MM cell lines at 24 hours after treatment with DMSO, 4-IPP, 10 μM ISO-1, Cfz, 4-IPP+Cfz, or ISO-1+Cfz. (C) Summarized results of apoptotic rates in KMS-11 or KMS-11/Cfz MM cells at 24 hours after treatment with DMSO, 4-IPP, and Cfz at the indicated concentrations, or 4-IPP+Cfz. Flow cytometry histogram (D) and summarized results (E) of apoptotic rates in primary MM cells (n = 6) treated with DMSO, 4-IPP, Cfz, or 4-IPP+Cfz for 24 hours. Mitochondrial respiration (OCR) (F) and summarized results of basal OCR, maximum OCR, spare OCR (G), or ATP production OCR (H) in ARP-1 MM cells treated with DMSO, 4-IPP, Cfz, or 4-IPP+Cfz for 16 hours. (I) Misfolded SOD1 expression in ARP-1 MM cells treated with DMSO, 4-IPP, Cfz, or 4-IPP+Cfz for 16 hours. (J) NSG mice were injected subcutaneously (s.c.) with 2 × 10⁶ CTR-KO or MIF-KO ARP-1 MM cells. At day 7 after tumor inoculation, vehicle (Veh) or 3 mg/kg Cfz were IV injected, 2 consecutive days in a week and repeated for 3 weeks, into MM-bearing mice (n = 5 for each group). At day 8, MM-bearing mice were intraperitoneally (IP) injected with L 012 sodium salt and detected in vivo for ROS signal by bioluminescent imaging (K,L). (M) Tumor volume was calculated from caliper measurements every 3 to 4 days. (N) Survival curves of CTR-KO or MIF-KO MM-bearing mice treated with Veh or Cfz. (O,P) NSG mice were injected s.c. with 2 × 10⁶ ARP-1 MM cells. At day 8 after tumor inoculation, Veh or 1 mg/kg Cfz was IV injected, 2 consecutive days weekly, into MM-bearing mice. At day 28 when the size of some tumors enlarged to 10 mm, MM-bearing mice received treatment with a high dose of Cfz (3 mg/kg for 2 consecutive days weekly; n = 6) or 4-IPP (0.5 mg per mouse for every 3 days; n = 7) alone, or their combinations (n = 6) until the end of the experiment. Tumor volume (O) and survival curves (P) were calculated. (Q-T) NSG mice were injected IV with 2 × 10⁶ MM.1S or ARP-1 MM cells. At day 14 after tumor inoculation, Veh, 4-IPP, Cfz, or 4-IPP+Cfz was injected into MM-bearing mice (n = 5 per group). Blood samples were collected weekly starting at day 16. Tumor burden (Q,S), analyzed by using enzyme-linked immunosorbent assay measuring human immunoglobulin light chain in mouse plasma which was normalized to control, and survival (R,T) in MM.1S or ARP-1 MM-bearing mice were calculated. The Student t test was used to compare 2 samples. Tumor burden was analyzed by one-way analysis of variance with Tukey’s post hoc test at each time point. The survival plots in panels N, P, R, and T show Kaplan-Meier estimates of survival and comparisons using the log-rank test.*P < .05; **P < .01; ***P < .001. n.s., not significant.

Figure 6. — MIF inhibitors sensitize human MM cells to PIs in vitro and in vivo. (A) Cell viability of human MM cell lines pulsed with DMSO or 80 nM Cfz for 1 hour and treated without or with 10 μM 4-IPP (4-IPP+Cfz) for 48 hours, normalized with DMSO-treated group. (B) Summarized results of apoptosis of human MM cell lines at 24 hours after treatment with DMSO, 4-IPP, 10 μM ISO-1, Cfz, 4-IPP+Cfz, or ISO-1+Cfz. (C) Summarized results of apoptotic rates in KMS-11 or KMS-11/Cfz MM cells at 24 hours after treatment with DMSO, 4-IPP, and Cfz at the indicated concentrations, or 4-IPP+Cfz. Flow cytometry histogram (D) and summarized results (E) of apoptotic rates in primary MM cells (n = 6) treated with DMSO, 4-IPP, Cfz, or 4-IPP+Cfz for 24 hours. Mitochondrial respiration (OCR) (F) and summarized results of basal OCR, maximum OCR, spare OCR (G), or ATP production OCR (H) in ARP-1 MM cells treated with DMSO, 4-IPP, Cfz, or 4-IPP+Cfz for 16 hours. (I) Misfolded SOD1 expression in ARP-1 MM cells treated with DMSO, 4-IPP, Cfz, or 4-IPP+Cfz for 16 hours. (J) NSG mice were injected subcutaneously (s.c.) with 2 × 10⁶ CTR-KO or MIF-KO ARP-1 MM cells. At day 7 after tumor inoculation, vehicle (Veh) or 3 mg/kg Cfz were IV injected, 2 consecutive days in a week and repeated for 3 weeks, into MM-bearing mice (n = 5 for each group). At day 8, MM-bearing mice were intraperitoneally (IP) injected with L 012 sodium salt and detected in vivo for ROS signal by bioluminescent imaging (K,L). (M) Tumor volume was calculated from caliper measurements every 3 to 4 days. (N) Survival curves of CTR-KO or MIF-KO MM-bearing mice treated with Veh or Cfz. (O,P) NSG mice were injected s.c. with 2 × 10⁶ ARP-1 MM cells. At day 8 after tumor inoculation, Veh or 1 mg/kg Cfz was IV injected, 2 consecutive days weekly, into MM-bearing mice. At day 28 when the size of some tumors enlarged to 10 mm, MM-bearing mice received treatment with a high dose of Cfz (3 mg/kg for 2 consecutive days weekly; n = 6) or 4-IPP (0.5 mg per mouse for every 3 days; n = 7) alone, or their combinations (n = 6) until the end of the experiment. Tumor volume (O) and survival curves (P) were calculated. (Q-T) NSG mice were injected IV with 2 × 10⁶ MM.1S or ARP-1 MM cells. At day 14 after tumor inoculation, Veh, 4-IPP, Cfz, or 4-IPP+Cfz was injected into MM-bearing mice (n = 5 per group). Blood samples were collected weekly starting at day 16. Tumor burden (Q,S), analyzed by using enzyme-linked immunosorbent assay measuring human immunoglobulin light chain in mouse plasma which was normalized to control, and survival (R,T) in MM.1S or ARP-1 MM-bearing mice were calculated. The Student t test was used to compare 2 samples. Tumor burden was analyzed by one-way analysis of variance with Tukey’s post hoc test at each time point. The survival plots in panels N, P, R, and T show Kaplan-Meier estimates of survival and comparisons using the log-rank test.*P < .05; **P < .01; ***P < .001. n.s., not significant.