Lenalidomide-mediated intracellular H2O2 elevation induces ER stress. (A) OPM2-NT and shCRBN cells were treated for 72 hours with lenalidomide (10 μM) or DMSO (control). Cell lysates were prepared, separated by electrophoresis, and immunoblotted with the indicated antibodies. (B) OPM2-NT and OPM2-shCRBN knockdown cells were incubated with lenalidomide (10 μM) for 48 hours. Secreted and intracellular IgL-λ levels were determined via enzyme-linked immunosorbent assay. Data are expressed as a percentage of control (mean ± standard deviation, n = 3). (C) OPM2-NT and OPM2-CRBN knockdown (shCRBN) cells were treated with lenalidomide for 3 days. Cell lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. (D) Lenalidomide induced XBP-1 mRNA splicing in CRBN-positive cells but showed minimal splicing in shCRBN cells. OPM2-NT and shCRBN cells were treated with lenalidomide (10 μM) for 3 days. Reverse transcription polymerase chain reaction was performed to evaluate XBP-1 mRNA splicing, a marker of ER stress. (E) OPM2-NT and shCRBN cells were treated for 6 days with lenalidomide (10 μM). Cell lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. Lenalidomide-induced, ER stress–mediated PARP degradation was more prominent at day 6 in OPM2-NT cells compared with shCRBN cells. (F) OCIMY5 cells overexpressing CRBN progressively accumulated IgL dimers after lenalidomide treatment and induced ER stress. OCIMY5-vector and CRBN cells were treated with lenalidomide (10 μM) for different periods (24-96 hours). Cell lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. (G) Lenalidomide-sensitive (MM.1S) and lenalidomide-resistant (MM.1S.res) cell lines were treated with lenalidomide (10 μM) for 3 days. Cell lysates were prepared and immunoblotted as indicated. (H) CRBN-expressing but with high antioxidative capacity exhibiting MM cell line, RPMI-8226, treated with lenalidomide (10 μM) for 3 days. Cell lysates were prepared 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.
