Figure 6
Comparison of lenalidomide and pomalidomide effects in DF15 and DF15R myeloma cells. (A) Dose-dependent inhibition of proliferation of parental DF15 cells by lenalidomide (•) and pomalidomide (▪). No proliferation inhibition of DF15R cells by lenalidomide (○) or pomalidomide (□). Insert is immunoblot analysis of protein lysates from DF15 and DF15R cells demonstrating minimal CRBN protein in DF15R compared with DF15 cells. (B) CRBN analysis in DF15 and DF15R cells by immunofluorescence confocal microscopy in the absence (panels a) or presence (panels b) of anti-CRBN blocking peptide (Supplemental Figure S2). Images were obtained using a Nikon E800 confocal microscope (Nikon Instruments, Melville, NY, USA) at a 60 × magnification. CRBN immunofluorescence is shown as green color and DAPI blue staining identifies the nucleus of cells. (C) Immunoblot of thalidomide analog affinity bead binding to CRBN in DF15 and DF15R cell extracts. Lane description in order left to right: In=DF15 input before bead purification; DMSO=DF15 extract control (1% DMSO preincubation); Len=DF15 extract preincubated with lenalidomide (100 μM); In=DF15R input before bead purification; DMSO=DF15R control (1% DMSO preincubation); Len=DF15R extract preincubated with lenalidomide (100 μM). Representative immunoblot from two independent experiments with similar results.
