Responses of ALL xenografts to dexamethasone in vitro. (A) Xenograft cells were retrieved from cryostorage, and metabolic activity was assessed after a 48-hour exposure to a serial dilution of dexamethasone (10−5-10−9) relative to vehicle-treated controls by MTT assay. (B) Protein expression of Bim was assessed by immunoblot analysis relative to Actin control in glucocorticoid-sensitive (ALL-3 and ALL-11) and -resistant (ALL-2 and ALL-19) xenograft cells after treatment with dexamethasone (1μM, 16 hours). (C) Gene expression of glucocorticoid responsive genes, FKBP5, SOCS1, and BIM, was analyzed by RT2-PCR and expressed as a fold of vehicle-treated controls after treatment with dexamethasone (1μM) for up to 16 hours. (D) ChIP was carried out to identify recruitment of RNA polymerase II to the TSS of the BIM locus, and Foxo3a to its binding site upstream of the TSS, with each PCR region evaluated relative to total histone H3 ChIP in the same sample. Results were visualized by polyacrylamide gel electrophoresis and quantified by SYBR-green PCR. Splicing within a representative gel is indicated by dividing spaces. Quantified results represent the mean ± SEM of 3 independent experiments.
