mTOR, a master negative regulator of autophagy, is an ATF5 target gene. (A) qRT-PCR analysis monitoring expression of 62 autophagy-related genes in 32D/BCR-ABL cells after treatment with an ATF5 siRNA. Expression of Atf5 is shown as a positive control. The expression of each gene was normalized to that obtained after treatment of cells with a luciferase siRNA. The red line indicates 2-fold decrease in expression. Error bars represent SD. (B) 32D/BCR-ABL cells treated with a NS or ATF5 shRNA were monitored for mTOR and phosphorylated mTOR levels by immunoblot analysis. (C) qRT-PCR analysis (left) or immunoblot analysis (right) monitoring mTOR expression in K562 cells treated with either a NS or ATF5 shRNA. Error bars represent SD. (D) ChIP analyses monitoring binding of ATF5 to the mTOR promoter in 32D/BCR-ABL cells in the presence or absence of imatinib (5μM for 24 hours). (E) Luciferase reporter assay. Fragments containing 539, 2010, 2017, or 3007 bp of the mTOR promoter, or as a control empty vector, were tested for their ability to drive heterologous firefly luciferase gene after transient transfection in 32D/BCR-ABL cells. Error bars represent SD. (F) 32D/BCR-ABL cells transfected with the reporter construct containing 3007 bp of the mTOR promoter were treated with either DMSO, imatinib (5μM for 24 hours), LY294002 (20μM for 24 hours), PHT-427 (20μM for 24 hours) or JAK inhibitor I (10μM for 24 hours). (G) qRT-PCR monitoring mTOR 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. *P < .05; **P > .05.