Figure 2.
Knockdown of MMSET in t(4;14)-positive MM causes resistance to ferroptosis. (A) Knockdown efficiency was analyzed by western blotting in t(4;14)-positive NCI-H929 and OPM-2 cells transfected with shRNAs against MMSET (shMMSET) and scramble shRNA as the negative control (shNC) for 48 hours. GAPDH was used as the endogenous control. (B) Showing the relative cell viability of NCI-H929 and OPM-2 cells with or without MMSET knockdown treated with RSL3 at indicated concentrations for 24 hours. The IC50 analysis was conducted using GraphPad software 8.0. (C) Relative cell death analysis of NCI-H929 and OPM-2 cells with or without MMSET knockdown that were pretreated with the indicated cell death inhibitors for 2 hours and then treated by RSL3 (0.5 μM for NCI-H929 and 0.2 μM for OPM-2) for 24 hour. (D-E) The relative PTGS2 mRNA levels (D) or MDA concentrations (E) of NCI-H929 and OPM-2 cells with or without MMSET knockdown with RSL3 treatment (0.5 μM for NCI-H929 and 0.2 μM for OPM-2) for 24 hours. (F) Lipid ROS levels of NCI-H929 and OPM-2 cells with or without MMSET knockdown, treated with RSL3 (0.5 μM for NCI-H929 and 0.2 μM for OPM-2) for 6 hours in the presence or absence of Fer-1. (G) Electron microscopy images of of NCI-H929 cells with or without MMSET knockdown treated with 0.5 μM RSL3 or DMSO. The black arrowhead indicates mitochondria with membrane rupture and reduced cristae in the group of shNC and relatively intact mitochondrial structures in the shMMSET group with RSL3 treatment. The levels of shrunken mitochondria were analyzed using ImageJ software. The left scale bar: 1 μm. The right scale bar: 0.5 μm. (H-I) Relative cell viability of NCI-H929 and OPM-2 cells with MMSET knockdown (H) or t(4;14)-negative U-266 cells (I) treated with RSL3 at the indicated concentrations for 24 hours in the presence of PUFAs (5 μM arachidonic acid or adrenic acid) or MUFAs (oleic acid) and followed by treatment with the ferroptosis inhibitor Fer-1 or not. ns, not significant; P > .05, ∗ P <0 .05, ∗∗ P < .01, and ∗∗∗ P < .001.

Knockdown of MMSET in t(4;14)-positive MM causes resistance to ferroptosis. (A) Knockdown efficiency was analyzed by western blotting in t(4;14)-positive NCI-H929 and OPM-2 cells transfected with shRNAs against MMSET (shMMSET) and scramble shRNA as the negative control (shNC) for 48 hours. GAPDH was used as the endogenous control. (B) Showing the relative cell viability of NCI-H929 and OPM-2 cells with or without MMSET knockdown treated with RSL3 at indicated concentrations for 24 hours. The IC50 analysis was conducted using GraphPad software 8.0. (C) Relative cell death analysis of NCI-H929 and OPM-2 cells with or without MMSET knockdown that were pretreated with the indicated cell death inhibitors for 2 hours and then treated by RSL3 (0.5 μM for NCI-H929 and 0.2 μM for OPM-2) for 24 hour. (D-E) The relative PTGS2 mRNA levels (D) or MDA concentrations (E) of NCI-H929 and OPM-2 cells with or without MMSET knockdown with RSL3 treatment (0.5 μM for NCI-H929 and 0.2 μM for OPM-2) for 24 hours. (F) Lipid ROS levels of NCI-H929 and OPM-2 cells with or without MMSET knockdown, treated with RSL3 (0.5 μM for NCI-H929 and 0.2 μM for OPM-2) for 6 hours in the presence or absence of Fer-1. (G) Electron microscopy images of of NCI-H929 cells with or without MMSET knockdown treated with 0.5 μM RSL3 or DMSO. The black arrowhead indicates mitochondria with membrane rupture and reduced cristae in the group of shNC and relatively intact mitochondrial structures in the shMMSET group with RSL3 treatment. The levels of shrunken mitochondria were analyzed using ImageJ software. The left scale bar: 1 μm. The right scale bar: 0.5 μm. (H-I) Relative cell viability of NCI-H929 and OPM-2 cells with MMSET knockdown (H) or t(4;14)-negative U-266 cells (I) treated with RSL3 at the indicated concentrations for 24 hours in the presence of PUFAs (5 μM arachidonic acid or adrenic acid) or MUFAs (oleic acid) and followed by treatment with the ferroptosis inhibitor Fer-1 or not. ns, not significant; P > .05, ∗ P <0 .05, ∗∗ P < .01, and ∗∗∗ P < .001.

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