Effect of NPI-0052 plus lenalidomide on neovascularization, ubiquitination, BIM, Bcl-6, and YB-1 in vivo in xenografted MM tumors. Tumor sections from mice receiving indicated treatment were immunostained with factor VIII (A), VEGFR1, or ubiquitin Abs (B). (C) Tumor lysates from control and drug-treated mice were subjected to immunoblot analysis using anti-BIM or antiactin Abs. Lanes 1 through 6 represent tumor lysates from mice receiving the following treatments: lane 1, vehicle alone (control); lane 2, NPI-0052 (0.15 mg/kg); lane 3, lenalidomide (2.5 mg/kg); lane 4, lenalidomide (5.0 mg/kg); lane 5, NPI-0052 (0.15 mg/kg) plus lenalidomide (2.5 mg/kg); and lane 6, NPI-0052 (0.15 mg/kg) plus lenalidomide (5.0 mg/kg). (D) Bar graph represents quantification of BIMEL protein bands in immunoblot shown in Figure 4C by densitometry: a 3.3-fold increase in BIMEL isoform was noted in NPI-0052 plus lenalidomide–treated versus untreated cells. Samples were normalized to actin. (E) Tumor sections from control and NPI-0052 plus lenalidomide–treated mice were immunostained with YB-1 Ab. (F) Tumor lysates from control and drug-treated mice were subjected to immunoblot analysis using anti–Bcl-6 or antiactin Abs. Lanes 1 through 6 are the same as in panel C. (A-B,E) Representative of similar observations in 2 different mice receiving the same treatment. For panels A, B, and E, images were obtained with a Zeiss Axioimager M1 microscope (63×/1.4 Plan-Apochromat objective), a AxioCam HRc camera, Axiovision Version 4.6 software, and permount imaging solution.
