Influence of ST1346 and ATRA on the levels and state of transcriptional factors or complexes involved in ATRA-induced granulocytic differentiation of myeloid cells.

NB4 cells were treated for 72 hours (panel A) or the indicated amount of time (panels B and C) with vehicle, ATRA (0.1 μM), the indicated concentrations of ST1346, or the corresponding combinations of the BISIND and the retinoid. (A,B) Equivalent amounts of cell extracts were subjected to Western blot analysis with anti–STAT 1 α/β, anti–β-actin, anti–CEBPε polyclonal antibodies, or antibodies recognizing the Tyr701 phosphorylated form of STAT 1. The right graph in panel A illustrates the quantitative results obtained by densitometric analysis of the STAT 1α band in blots similar to that depicted on the left and developed with an anti–STAT 1α–specific antibody. The levels of STAT 1α protein are expressed in relative units following normalization for the intensity of the corresponding β-actin bands. Data are expressed as the means ± SD of 3 separate experiments. *Significantly higher than the corresponding group treated with vehicle (P < .01 according to the Student t test). °Significantly higher than the sum of the effects observed in the corresponding ST1346- and ATRA-treated groups, following 2-way analysis of variance and measurement of the F score of the interaction (P < .01 according to Tukey test). (C) Cell extracts were subjected to gel retardation assays using double-stranded oligonucleotides corresponding to the consensus sites of binding of the transcriptional complexes Ets-1 and CREB. The competitor is a cold Ets-1 (ets) or CREB oligonucleotide present in the reaction mixture in 100-fold excess.