AML-1/ETO antagonizes PLZF-mediated transcriptional repression in a dominant-negative fashion.

Transient transfections and reporter assays were all performed in 293T cells. (A) 293T cells transfected with the IL-3Rα reporter construct. Lanes 1-8, (12-well dishes): 400 ng each of pCDNA expression vector, pCDNA-PLZF, pCMV expression vector, and pCMV-AML-1/ETO was transfected as indicated. Results are expressed as fold repression compared to the empty expression vector. (B) Transient transfections performed in conditions similar to those described for A. In this case the results are expressed as relative luciferase activity compared to vector alone. (C) 6-well dishes: 800 ng pCDNA or pCDNA-PLZF was cotransfected with increasing doses of AML-1/ETO as follows: lanes 2 and 8, 100 ng; lanes 3 and 9, 200 ng; lanes 4 and 10, 400 ng; lanes 5 and 11, 800 ng; lanes 6 and 12, 1200 ng AML-1/ETO, respectively. (D) 293T cells were plated at a density of 2.8 × 10⁵ in 6-well dishes and transfected with 800 ng pCDNA, PLZF, or AML-1/ETO as indicated and identical amounts of reporter and internal controls, as in C. The cells were harvested simultaneously with those used for luciferase assays and were counted. (E) 293T cells transfected with the (GAL4)₅-tk-Luc reporter. Lane 1, GAL4^1–147 (400 ng) transfected without AML-1/ETO. Lanes 2 and 3, GAL4^1–147 400 ng with AML-1/ETO 200 ng and 400 ng, respectively. Lane 4, GAL4-KRIP1 (400 ng). Lanes 5 and 6, GAL4-KRIP1 (400 ng) along with AML-1/ETO 200 ng and 400 ng, respectively. (C, D, E) Fold repression was compared to vector only. The results of all graphs reflect the average of multiple experiments ± SEM.