Thalidomide and CC-4047 do not affect terminal differentiation of erythroid progenitor cells. SCF/Epo erythroid progenitor cells were generated from SI2 cells after 3 to 4 days of culture with SCF plus Epo plus Dex as indicated in Figure 2A. Then, 3 × 10⁶ SCF/Epo cells were induced to terminal differentiation with Epo plus insulin in the presence of thalidomide (100 μM), CC-4047 (100 μM), CC-5013 (10 μM), or 0.1% DMSO (control). (A) The growth kinetics are shown as cumulative cell numbers. Data represent means ± standard deviation from 3 different experiments. □ indicates thalidomide; ▪, CC-4047; and × with dotted line, control. (B) Cell hemoglobin levels were determined by hemoglobin assay and measured as optical density (OD 490). After 0, 2, and 3 days in the presence of thalidomide (, 100 μM), CC-4047 (▪, 100 μM), or 0.1% DMSO (□, control). Data represent means ± standard deviation. (C) At day 2 in culture, cells were subjected to apoptosis assay by double staining with annexin V. Result is shown as the ratio of living cells (▪), apoptotic cells (▨), and necrotic cells (□) in the total cell numbers. (D) Cells at day 3 in culture. Cells were analyzed for surface antigen expression by flow cytometry (filled area). The open area indicates staining with isotype control antibody. Horizontal bars indicate the percent of positive cells for each staining.