Lenalidomide inhibits intracellular H₂O₂ decomposition in MM cells. (A) MM.1S cell line was pretreated with Amplex Red (50 μM) and incubated with increasing concentrations of lenalidomide (0.625-10 μM) or dimethyl sulfoxide (DMSO; control) for 90 minutes before fluorescence intensity was measured. (B) IMiDs inhibit intracellular H₂O₂ decomposition in HMCLs. Cells were pretreated with Amplex Red (50 µM) and incubated with thalidomide (20 µM), lenalidomide (20 µM), pomalidomide (10 µM), or DMSO (control) for 40 to 90 minutes. All IMiDs inhibited intracellular H₂O₂ decomposition in HMCLs. (C) IMiDs inhibit HRP-mediated decomposition of H₂O₂ in an in vitro assay. Thalidomide, lenalidomide, or pomalidomide (10 µM for all) or DMSO (control) was incubated with HRP, Amplex Red, and H₂O₂ (5 µM) for 30 minutes before fluorescence intensity was measured. (D) Lenalidomide inhibits intracellular H₂O₂ decomposition in samples from patients with primary MM. CD138⁺ samples were pretreated with Amplex Red (50 μM) and incubated with lenalidomide (1 or 10 μM) or DMSO (control) for 90 minutes. Data are shown as mean ± SEM, n = 4 biological repeats. (E) Lenalidomide-sensitive (MM.1S) and lenalidomide-resistant (MM.1S.res) cell lines were treated with lenalidomide for 3 days. Cell lysates were prepared under nonreducing conditions; protein lysates were separated by electrophoresis under nonreducing conditions (without DTT) and immunoblotted as indicated. (F) MM.1S and MM.1S.res cells were seeded and incubated with lenalidomide at the indicated concentration for 3 days, and MTT assays were performed. Each experimental condition was performed in triplicate and repeated at least twice. (G-H) CRBN-overexpressing OCIMY5 and vector control cells and CRBN-knockdown OPM2 cell lines were treated with lenalidomide for 3 days. Cell lysates were both prepared and separated by electrophoresis under nonreducing conditions (without DTT) to detect IgL dimerization. Immunoblots were performed as indicated. Blots are representative of 3 independent experiments. (I-J) CRBN-positive cells were more sensitive to lenalidomide than CRBN-negative cells, as measured by the MTT assay. Each experimental condition was performed in triplicate and repeated at least twice. All data are shown as mean ± SEM for a minimum of 3 independent experiments. *P < .05 compared with DMSO (control); **P < .01 compared with control; ***P < .001 compared with control.