Leukemic cells from a patient with APL (HT93 cells) have suppressed PU.1 expression. (A) Northern blot analysis using a PU.1 probe showing myeloid U937 cells lacking the PML-RARA translocation (first lane), t(15;17) HT93A cells (second lane), and uninduced U937-PR9 cells (third lane). The somewhat lower PU.1 expression in U937-PR9 versus U937 cells may be due to leakiness of the system and thus low level expression of PML-RARA in the absence of zinc. The blot was reprobed with a GAPDH probe. (B) Prolonged exposure of a Western analysis using a PU.1 antiserum (top). U937 cells served as a positive control. The blot was reprobed with a β-tubulin antibody (bottom). (C) EMSA studying the binding activity to the PU.1 site in the M-CSF receptor promoter. U937 cells (U9; lanes 4 and 6) were compared with HT93A cells (HT; lanes 3 and 5). ivt indicates in vitro-translated PU.1 protein is run as a control; P, free probe alone; SS, supershift with specific antibody; PU.1, shift of PU.1 protein binding to this particular site; and X, nonspecific binding.