ATRA-induced differentiation of AML cells downregulates SP1 promoter binding and CD95 expression. (A) Assessment of HL60 and NB4 ATRA-induced differentiation by NBT reduction assay. Cells were treated with 10μM ATRA or vehicle only (DMSO) for 48 hours. After treatment, cells were resuspended in 3 × 10−7M phorbol 12-myristate 13-acetate diluted in 1 mg/mL NBT solution. Cytospins were prepared from 100 μL of each cell suspension, with blue staining in ATRA-treated cells indicative of phagocytic capability and therefore confirming differentiation. (B-C) EMSA illustrating down-regulation of SP1 binding to the polymorphic site in the CD95 core promoter after ATRA treatment. (B) 32P-labeled CD95 −1377 DNA probe (G allele, all lanes) was incubated with 15 μg HL60 or NB4 nuclear extract from cells treated with 10μM ATRA or vehicle alone (DMSO). (C) SP1 binding was quantified using densitometry. Error bars represent the mean ± SEM from 3 independent experiments, and statistical analysis was performed using the unpaired Student t test. (D-E) ATRA-induced differentiation of HL60 and NB4 down-regulates SP1 and CD95 expression. HL60 or NB4 cells were incubated with 10μM ATRA or vehicle-alone (DMSO). Down-regulation of SP1 and CD95 protein expression was confirmed by immunoblot analysis (D) and quantified using densitometry (E). Error bars represent the mean ± SEM from at least 3 independent experiments, and statistical analysis was performed using the unpaired Student t test (E).
