Reduction of PU.1 expression by siRNA blocks ATRA-induced differentiation of HT93A cells. (A) HT93A cells were stably transfected with an MSCV retrovirus expressing PU.1 siRNA to reduce PU.1 expression (siPU.1-1 and siPU.1-2). Two pools of cells were expanded and analyzed after 1 month. Western blot for PU.1 demonstrates that treatment with 1 μM ATRA induced only small amounts of PU.1 protein in siPU.1 cells after 2 days compared with vector-transfected siV cells. (B) siPU.1 cells (pools siPU.1-1 and siPU.1-2) were induced with 1 μMATRAfor 5 days. FACS analysis was performed at day 0 (d0) and day 5 (d5) using a G-CSF receptor antibody. Parental HT93A cells transfected with the siRNA vector (siV) served as a positive control (right panel). (C) Wright-Giemsa staining of siPU.1 cells expressing PU.1 siRNA is shown before and after 5 days of ATRA treatment, indicating that siPU.1 cells morphologically fail to respond to ATRA treatment. Right panel: ATRA treatment induces neutrophil differentiation in the siV vector-transfected HT93A cells. (D) HT93A cells were transiently transfected with siRNAs against OCT-1, CEBPA, CEBPB, or CEBPE. PU.1 mRNA expression was determined by RT-PCR after 48 hours of ATRA treatment (and transfection). V indicates the silencer negative control siRNA no. 2 (Ambion) was used to control for nonspecific effects in siRNAexperiments.