Figure 3.

I3MO sensitizes MM cells to bortezomib-induced apoptosis. (a) The apoptosis of ARP1, U266, RPMI8226, ANBL6 and ANBL6 BR cells with monotherapy or combination therapy of BTZ (1.25,2.5,5,10 nM) or I3MO (1.25,2.5,5,10 µM) were detected after the treatment for 24 h by flow cytometry, respectively. Isobologram analysis shows the synergistic cytotoxic effect of BTZ and I3MO. CI value<1 indicates synergistic interactions. The experiments were performed in triplicate. (b) Xenograft mouse model of MM was utilized to detect the I3MO effects on MM cell growth. There were six treatment groups with varying dosage of I3MO and BTZ as indicated. (P<0.05, Two-way ANOVA). (c) Primary patient samples (n=5) derived xenograft model was further utilized to detect the cytotoxicity of I3MO in zebrafish. Schematic diagram of experimental design. (d) Patient-derived MM cells were labeled with Calcein-AM and implanted into the perivitelline space of each zebrafish. The survival of engrafted cells was monitored at 0 hpi and 24 hpi. Fluorescent microscopy imaging of Calcein-AM-labeled patient MM cells in zebrafish embryos treated with DMSO, BTZ, I3MO, or BTZ and I3MO, at 0 and 24hpi, respectively. Dashed lines circle the primary MM cells. (Scale bars: 100 μm.) (e) Quantification of Calcein-AM-positive (green) areas in zebrafish embryos at 24 h post-injection (5 patients/treatment) (P<0.05, t test). Data are mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, unpaired t test. MACS, magnetic cell sorting; MM cells, multiple myeloma cells; hpi, hours post-injection.