Figure 1. PML/RARA DNA-binding defective mutants failed to bind and activate RARE.

(A) Schematic diagram of P/R wild type (WT) and mutants used in the study. B, C, D, E and F, RARA functional regions; DBD, DNA-binding domain. (B) RARE3-tk Luc reporter plasmid and indicated protein expression constructs were transiently cotransfected into 293T cells. Renilla luciferase plasmid was transfected into cells as internal control. 14 hours after ATRA (10⁻⁶ M) or ethanol vehicle treatment, transactivation was measured. The number refers to fold of transactivity after ATRA treatment. Mean values were obtained from 3 independent experiments with duplicate wells. Error bars indicate standard deviations of triplicate measurements. *p<0.01. (C) Western blotting analysis of wild type and various mutant PML/RARA proteins with anti-RARA in transiently transfected 293T cells. (D) ChIP analysis of wild type or mutant PML/RARA binding to the endogenous RARB promoter in transfected 293T cells, expressed as increase over nonspecific binding. Means ± standard deviation (SD).