Figure 4.

IMiD compounds down-regulate C/EBPβ by targeting its protein translation. (A) MM.1S cells were cultured with pomalidomide, lenalidomide, or 0.01% DMSO (control). Total RNA was extracted from 12-hour cultures, reverse transcribed to cDNA, and used for quantitative real-time PCR. Data were analyzed according to the ΔΔC_t method. Results are shown as mRNA expression relative to control (DMSO). mRNA levels were normalized with β-actin mRNA expression as control. (B) MM.1S cells were incubated with pomalidomide (10μM) with and without cycloheximide (20 μg/mL) for 12, 24, or 36 hours. Cells were lysed, and whole cell lysates were analyzed by Western blotting for C/EBPβ. (C) MM.1S, H929, OPM2, or primary myeloma cells were cultured with lenalidomide, pomalidomide, or 0.01% DMSO as control for 3 days. Cells were lysed, and whole cell lysates were analyzed by Western blotting for eIF4E. β-Actin expression was probed for loading control. (D-E) MM.1S cells (2 × 10⁶) were incubated with lenalidomide (D) or pomalidomide (E) at different concentrations with a 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 whole cell lysates were analyzed for eIF4E expression by Western blotting. β-Actin expression was probed for loading control. (F) MM.1S cells were incubated with lenalidomide or pomalidomide for 12 hours, and total RNA was extracted by Trizol and followed by real-time PCR. Data were analyzed according to the ΔΔC_t method. Results are shown as mRNA fold compared with control (DMSO). (G) eIF4E knockdown cell lines were generated by lentiviral infection of MM.1S cells. 1# and 2# indicate different eIF4E shRNA sequences. Green fluorescence protein was used as control for eIF4E shRN-expressing cells. Cell lysates were analyzed by Western blotting to compare the levels of eIF4E, C/EBPβ, and IRF4. β-actin expression was probed for loading control.