Figure 6. C/EBPβ is required for ALK-mediated transformation and tumor growth.

(A and B) Downregulation of C/EBPβ expression by RNAi. TS cells were transduced with lentivirus (pLKO-GFP) expressing control (4C) or 4 C/EBPβ-directed shRNAs (10A–D). C/EBPβ mRNA and protein expression were determined by RT-PCR (A) and Western blot (B) analyses (96 hours). As a control, β2-microglobulin mRNA, α-tubulin, and phospho-eIF2α protein expression was documented. (C and D) C/EBPβ knock down compromises viability of ALCL cells in vitro. (C) TS cells were transduced with the indicated shRNA lentivirus. Percentage of apoptotic cells (sub-G₀/G₁) was determined by PI staining at day 5. (D) ALK-positive ALCL (TS, Su-DHL1, and JB) and ALK-negative (MAC-1 and K-562) cell lines were transduced with C/EBPβ (10D) or mock shRNA constructs. Apoptotic cells were determined at day 7. These findings are representative of 3 independent experiments. Error bars indicate SD. (E and F) C/EBPβ expression is required for tumor growth of ALK-positive cells in vivo. WT and c/ebpb^–/– MEFs were transduced with a retrovirus expressing EGFP/NPM-ALK. NPM-ALK, C/EBPβ, and α-tubulin protein expression was determined by Western blotting (E) prior to subcutaneous inoculation (5 × 10⁵ cells/mouse) into 8 athymic nu/nu mice recipients. Tumor growth was monitored over time (F). Error bars indicate SD.