Activity of thalidomide and CC-4047 on development of late-stage erythroid progenitor cells. (A) SI2 cells were generated from CD34+ cells after 7 days of culture with SCF plus IL-3 plus hyper–IL-6 as indicated in Figure 2A. Then, 3 × 106 SI2 cells were induced to differentiate into SCF/Epo cells with SCF, Epo, and Dex in the presence of thalidomide (10, 100 μM), CC-4047 (10, 100 μM), or 0.1% DMSO (control). At day 4 in culture, cells were analyzed for surface antigen expression by flow cytometry (filled area). The open area indicates staining with isotype control antibody. (B) The growth kinetics of cells from panel A are shown as cumulative cell numbers. ○ and • indicate 10 μM thalidomide and CC-4047, respectively; □and ▪, 100 μM thalidomide and CC-4047, respectively. × with dotted line indicates control. (C) At day 2 in culture, cells were subjected to apoptosis assay by double staining with annexin V and PI. Results are shown as the ratio of living cells (▪), apoptotic cells (▨), and necrotic cells (□) in the total cell numbers.
