H₂O₂ leads to preferential degradation of IKZF1 and IKZF3 in CRBN-positive cells. (A) Degradation of IKZF1 and IKZF3 was observed only with lenalidomide and H₂O₂. MM.1S cell line was treated with lenalidomide (10 μM), etoposide (1 μM), doxorubicin (0.5 μM), H₂O₂ (100 μM), and dexamethasone (20 μM) for 3 hours. Cell lysates were prepared, separated by electrophoresis, and immunoblotted with the indicated antibodies. (B) Different HMCLs were infected with AdIKLuc or the control vector (AdLuc), and sufficient luciferase activity was confirmed. Cell lines were treated with increasing concentrations of H₂O₂ (100 or 500 µM) or lenalidomide (2 µM) for 1 hour, and luciferase activity was measured. Data are shown as mean ± SEM, n = 4 biological repeats. (C) To evaluate the role of CRBN on IKZF1 and IKZF3 degradation, OPM2-nontarget (control) and OPM2-shCRBN (silenced) cells were treated with lenalidomide (10 μM) or increasing concentrations of H₂O₂ (25 or 50 μM) for 3 hours. Protein lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. (D-E) To differentially evaluate the effect of H₂O₂ and lenalidomide on cell viability based on the level of CRBN expression, OPM2-NT and OPM2-shCRBN were treated with increasing concentrations of H₂O₂ and lenalidomide for 3 days. MTT assays were performed, and cell survival was plotted. Each experimental condition was performed in triplicate and repeated at least twice. (F) OCIMY-5-vector cells and an isogenic CRBN-overexpressing cell line were treated with lenalidomide (10 μM) or H₂O₂ (50 μM) for 3 hours. Protein lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. (G-H) OCIMY-5 cells overexpressing CRBN and control vector were treated with increasing concentrations of H₂O₂ and lenalidomide for 3 days. MTT assays were performed, and cell survival was plotted. Each experimental condition was performed in triplicate and repeated at least twice. (I) Lenalidomide inhibits TrxR activity in vitro. Rat liver TrxR was treated with lenalidomide (1 µM and 10 µM) or DMSO control, and absorbance was measured at 405 nM. Lenalidomide-treated samples show decreased absorbance compared with control (DMSO). Data are shown as mean ± SEM, n = 3 biological repeats. (J) MM.1S cell line treated with lenalidomide (10 µM), aurothiomalate (10 µM), or DMSO (control) for 3 hours. Protein lysates were prepared and blotted with indicated antibodies. (K) MM.1S and OPM2 cells were treated with lenalidomide (10 µM), PX12 (20 µM), or DMSO (control) for 3 hours. Protein lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. Blots are representative of 3 independent experiments. All data are shown as mean ± SEM for a minimum of 3 independent experiments. **P < .01 compared with control; ***P < .001 compared with control.