Influence of ST1346 and ATRA on the transcriptional activity of RARα in transfected COS-7 cells and on the levels of PML-RARα, RARα, and RXRα proteins as well as histone acetylation in NB4 cells.

(A) COS-7 cells were transfected with the plasmid pSG5 (void vector) and the same plasmid containing RARα or RXRα as indicated. The transfection mixture also contained the retinoid-responsive reporter construct TRE-TK-Luc (containing the luciferase reporter gene) and the β-galactosidase expression vector pCH110. Twenty-four hours following transfection, cells were treated with vehicle (dimethyl sulfoxide [DMSO]), ST1346 (5 μM), ATRA (0.1 μM), or the combination of the 2 compounds for a further 24 hours. Cell extracts were used for the determination of luciferase enzymatic activity. The results are always normalized for the transfection efficiency as assessed by determination of the β-galactosidase enzymatic activity. Results are expressed as the means ± SD of 3 separate dishes and are representative of at least 3 experiments. (B) NB4 cells were treated for 72 hours with vehicle, ATRA (0.1 μM), and ST1346 at the indicated concentrations, or combinations of ATRA + ST1346. Aliquots of cell extracts were subjected to Western blot analysis with anti-RARα, anti-RXRα, and anti–β-actin antibodies. The positions of relevant molecular weight markers are indicated on the right. (●) indicates the undegraded form; * represents a degradation band of PML-RARα. (C) NB4 cells were treated for 72 hours with vehicle, ST1346 (5 μM), sodium butyrate (1 mM), and ATRA at the indicated concentrations or combinations of ATRA + ST1346 and ATRA + sodium butyrate. Aliquots of cell extracts were subjected to Western blot analysis with antibodies recognizing histone H3 or the acetylated form of histone H3 (Ac-H3). Results are representative of 2 independent experiments.