Conditional expression of PML-RARA in myeloid cells suppresses PU.1. (A) Top: U937 cells stably transfected with the PML-RARA cDNA (U937-PR9) under the control of the zinc-inducible metallothionein promoter were analyzed by Northern blot for PU.1 mRNA expression before and after treatment with 100 μM zinc. Bottom: The same blot was hybridized with a GAPDH probe. Zn indicates days of zinc treatment. (B) Western blots using a RARA antibody (top), a PU.1 antibody (middle), and a β-tubulin antibody (bottom). The PML-RARA protein was induced by treating U937-PR9 cells with 100 μM zinc. RARA indicates the endogenous RARA protein as present in U937 cells. Right panel: U937 cells lacking the zinc-inducible construct were treated with zinc to exclude nonspecific effects of zinc. (C) EMSA of binding activity to the PU.1 site in the M-CSF receptor promoter (left panel). U937-PR9 cells were induced with zinc. X represents nonspecific binding commonly observed with this oligonucleotide; ivt; in vitro-translated PU.1 protein is run as a control; P, free probe alone; Cos, nuclear extracts from Cos cells serve as a negative control; SS, supershift with specific antibody; and PU.1, shift of PU.1 protein binding to this particular site. The right panel represents binding of the same extracts to an NFY site as a control for loading and integrity of the extracts. C indicates competition with cold oligo.