BCR-ABL stimulates ATF5 transcription through PI3K/AKT/FOXO4 signaling. (A) qRT-PCR monitoring ATF5 expression in 32D/BCR-ABL or K562 cells treated in the absence of presence of the PI3K inhibitor LY294002 (20μM for 48-72 hours). Error bars represent SD. (B) qRT-PCR monitoring ATF5 expression in 32D/BCR-ABL or K562 cells expressing either empty vector or a vector encoding PIK3CA(E545K). Error bars represent SD. (C) Luciferase reporter assays. A 1548-bp Atf5 promoter fragment, or as a control empty vector, was tested for its ability to drive a heterologous firefly luciferase gene after transient transfection in 32D/BCR-ABL cells. Error bars represent SD. (D) Luciferase reporter assays. 32D/BCR-ABL cells transfected with the reporter construct containing 1548 bp of the Atf5 promoter were treated with either DMSO, imatinib (5μM for 24 hours), LY294002 (20μM for 48 hours), PHT-427 (20μM for 24 hours), or JAK inhibitor I (10μM for 24 hours). Error bars represent SD. (E) qRT-PCR monitoring Atf5 expression in 32D/BCR-ABL cells stably expressing either a NS or FOXO4 shRNA and treated in the absence or presence of LY294002 (20μM for 18 hours). Error bars represent SD. (F) Luciferase reporter assay. 32D/BCR-ABL cells transfected with the reporter construct containing 1548 bp of the Atf5 promoter and expressing either an NS or FOXO4 shRNA were treated in the absence of presence of LY294002 (20μM for 18 hours). Error bars represent SD. *P < .05; **P > .05.