CREB shRNA induces apoptosis in HSCs and bone marrow progenitor cells. (A) Transduced human peripheral blood cells plated in methylcellulose for 3 weeks and stained with monoclonal antibodies for CD34 and CD38 expression. Cells were stained with PI to assess cell death. (B) Murine bone marrow cells were transduced at a density of 10⁶ cells/mL with lentivirus expressing CREB, scrambled, or vector shRNA at a multiplicity of infection (MOI) of 100. After 2 days of culturing in media containing cytokines (mIL-3, 10ng/mL; mSCF, 25 ng/mL; and hIL-6, 10 ng/mL), cells were sorted using flow cytometry for GFP expression. Sorted calls were cultured in cytokine containing media for 5 days and stained for annexin-V and PI. All experiments were performed in triplicate. (C) Western blot analysis with lysates from mouse BM cells (10⁶) transduced with CREB shRNA, scrambled, luciferase, and vector control lentivirus. Immunoblots were probed with anti-PARP or β-tubulin antisera. Error bars in panels A and B represent SE.