CREB is essential for leukemia cell proliferation and survival. (A) Human (K562, TF-1) leukemia cells were transduced with a lentivirus expressing no shRNA,¹ CREB shRNA-1,² CREB shRNA-2,³  or luciferase shRNA⁴  at a multiplicity of infection (MOI) of approximately 100. Wild-type cells⁵  were also used as a control. Western blot analyses were performed with CREB, phospho-CREB, and β-tubulin antisera. (B) Five micrograms of total RNA were extracted from transduced leukemia cells, and q-PCR was performed to determine CREB expression. CREB was knocked down by up to 75% relative to control shRNA (vector) in human myeloid leukemia cells. (C) Trypan blue exclusion method was performed in triplicate to assess growth and survival of transduced leukemia cells. CREB knocked-down cells demonstrated diminished proliferation and viability 72 hours after transduction. (D) K562 cells were transduced and cultured for 48 hours before harvesting total RNA. Parental K562 cells were cultured in the presence of interferon-2α (100 units/mL) for 48 hours as a positive control. Quantitative reverse transcription-PCR was performed in triplicate with primers specific for CREB, actin, and OAS-1. (E) Luciferase reporter assays in human TF-1 leukemia cells transduced with CREB or control shRNAs. Decreased transcriptional activity was observed in CREB knocked-down cells and repeated in triplicate. (F) Cell-cycle analysis of CREB knocked-down TF-1 cells after synchronization by serum starvation overnight and stimulated for 12 hours with GM-CSF revealed decreased percentage of cells in S-phase. Experiment was performed in triplicate. Error bars in panels B-F represent SE.