PU.1-ER(T) induces MPO RNA, but does not induce terminal granulopoiesis in 32D cl3 myeloblasts. (A) Ten micrograms of pB4TKCAT was transfected into NIH 3T3 cells with 10 μg pCMV-Pip and 10 μg of pPGK-PU.1 or pPGK-PU.1-ER(T) and was cultured with or without 100 nmol/L 4HT (lanes 1 through 4). pB4TKCAT was similarly transfected with pCMV-Pip and pPGK-AS-PU.1, which expresses an antisense PU.1 RNA, and cultured with 4HT (lane 5). CAT assays performed 48 hours after transfection are shown. (B) Total cellular protein extracts prepared from 32D cl3 cells and from two puromycin-resistant subclones transduced with the BabePuro-PU.1-ER(T) retroviral vector were subjected to Western blotting using an ER antisera. The location of PU.1-ER(T) is indicated. (C) Cytospins were prepared from 32D-PU.1-ER(T) cells in IL-3, in IL-3 and after exposure to 4HT for 14 days, and after transfer from IL-3– to G-CSF–containing media for 5 days. The cytospins were subjected to Wright’s-Giemsa staining. (D) Total cellular RNAs were prepared from 32D-PU.1-ER(T) cells in IL-3 after exposure to 4HT for 0, 1, or 2 days. These RNAs were subjected to Northern blotting for MPO RNA (top panel). The integrity of the RNA samples was assessed by ethidium bromide staining (bottom panel).