Effect of UBE2I-dominant-negative mutant on growth, proliferation, and adhesion to BMSCs. (A) Myc-tagged fusion proteins were expressed in RPMI 8226 cells. Mo indicates mock transfected; Wt, UBE2I wild-type; and DN, UBE2I-DN (dominant negative). Lysates were probed with a Myc-tag specific mAb (top), Ube2I-specific mAb (middle), or Sumo-1 polyclonal Ab (bottom). (B) UBE2I-DN increased γ-radiation–induced apoptosis. Early apoptosis was detected by annexin V staining. Plots indicate percentage of RPMI 8226 cells in early apoptosis 24 hours after treatment with 10 Gy γ-radiation. Data are representative of 3 independent experiments. (C) UBE2I-wt and UBE2I-DN resulted in opposite effects on MM cell growth. Values represent the mean of triplicate measurements using the MTT assay. (D) Ube2I is necessary for MM adhesion to bone marrow stroma. Adhesion of GFP-labeled UBE2I transfectants to normal BMSCs is shown by fluorescence microscopy after 96 hours of incubation under standard conditions. Data are representative of 3 independent experiments. Images were viewed with a Zeiss Axiovert 200 inverted epifluorescence microscope using a 20× objective (37°C; cells were in PBS; FITC fluorescent filter). Images were acquired with a Zeiss AxioCam HRc 14-bit color CCD camera and were processed with Axio Vision software (Version 3.1). (E) UBE2I-DN decreased BMSCs-induced uptake of 3H-thymidine. Values represent the mean of triplicate measurements of 3H-thymidine after 96 hours of coculture.
