IMiD compounds down-regulate C/EBPβ in MM cells in a time- and dose-dependent manner. (A) MM.1S and OPM2 cells (6 × 104/well) were cultured with lenalidomide or pomalidomide at different concentrations for 2 days. DMSO (0.01%) was used as the control treatment. DNA synthesis was measured by 3H-thymidine incorporation, and the amount of DNA synthesis in treated cells relative to the amount in control cells (as a percentage) was plotted. Results are shown as triplicates of mean ± SD. (B) MM.1S, H929, OPM2, or primary myeloma cells were cultured with DMSO, pomalidomide, or lenalidomide at the indicated concentrations for 3 days. Representative results from 3 independent experiments are shown. Cells were then lysed, and cell lysates were analyzed for C/EBPβ expression by Western blotting. β-actin expression was probed for loading control. The ratio of LAP/LIP is measured using ImageJ 1.44 software. (C-D) MM.1S cells were incubated with lenalidomide (C) or pomalidomide (D) at different concentrations with fixed time period of 4 days, or for different time periods with fixed concentration of 10μM, or with DMSO 0.01% as control. Cells were lysed and cell lysates were analyzed for C/EBPβ expression by Western blotting. β-Actin expression was probed for loading control.
