Apoptosis and proliferation levels in differentiating SBDS-deficient cells. (A) Control and knockdown K562 cells were induced to undergo erythroid differentiation for 5 days. Cells were fixed in 70% ethanol and stained with propidium iodide for DNA content analysis. A representative diagram of 4 independent experiments is shown. Pre-G₀/G₁ cells represent apoptotic cells. Representative distributions of the cell cycle phases are depicted on day 0 of wild-type cells. (B) Cells were costained for propidium iodide to evaluate pre-G₀/G₁ apoptotic cells and for Ki-67 expression on nonapoptotic cells to evaluate proliferating cells. Cells were then analyzed by flow cytometry. Comparison of the mean fold increase in apoptosis versus mean fold decrease in proliferation of 4 independent experiments is shown. The values for apoptosis and proliferation were normalized to the value obtained for each day in control K562 cells, and assigned a value of 1.0 for proliferating cells and apoptotic cells. (C) Mobilized CD34⁺ cells from 2 donors for hematopoietic stem cell transplantation were purified, transduced, and induced to undergo erythroid differentiation as described in “Cell culture and erythroid differentiation.” After 5 days, cells were harvested and evaluated for apoptosis rates using annexin V staining. For each donor cell (N1 and N2), annexin V staining of cells transduced with scrambled RNA controls (SCR) or shRNA against SBDS (S-3) are shown. (D) Results of inhibition of SBDS mRNA in CD34⁺ cell samples are shown. The faster running SBDS band corresponds to a previously published shorter isoform. (Cell numbers did not allow corresponding testing of SBDS protein levels.)