Lenalidomide- and bortezomib-mediated oxidative stress in MM cell death. (A) OPM2 cells treated with lenalidomide (2 μM), bortezomib (2.5 nM), or lenalidomide plus bortezomib for 3 days. Cell lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. (B) OPM2 cells, with or without 2 days of pretreatment with lenalidomide (10 μM), were treated with bortezomib (2.5 nM), lenalidomide (10 μM), and bortezomib plus lenalidomide for an additional 2 days. Samples were collected and stained with Annexin V-FITC and propidium iodide. Fluorescence-activated cell sorting (FACS) analysis for apoptosis showed the lowest viability with lenalidomide pretreatment followed by bortezomib plus lenalidomide. (C) Pretreatment with lenalidomide enhances bortezomib-mediated apoptosis in CRBN-positive MM cells. OPM2-NT and OPM2-shCRBN cells, with or without pretreatment with lenalidomide for 2 days, were treated with bortezomib (2.5 nM) for an additional 2 days. Cell lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. Lenalidomide pretreatment enhanced bortezomib-induced apoptosis of CRBN-positive cells but had a less pronounced effect in CRBN-knockdown cells. (D) OPM2 cells were treated with lenalidomide (10 µM) or with increasing concentrations of pomalidomide (0.5 and 2 μM) for 3 days. Cell lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. Pre, pretreatment with.