AML1-ETO regulates the cell cycle inhibitor p21^WAF1. (A) K562 cells infected with MigR1 and Mig-A/E were sorted by flow cytometry 2 days after infection for EGFP⁺ cells and cultured overnight at 2 × 10⁵ cells/mL and harvested for cell cycle analysis by propidium iodide staining by flow cytometry. Percentages of G₀/G₁ and cells in S/G₂/M phase of cells are presented from 3 individual infection experiments. (B) Following infection and flow cytometry sorting were analyzed by Western transfer with 20 μg protein the blots were probed with anti-HA or anti-p21waf1 to analyze their expression. Ponceau staining of the blot depicts loading. (C) K562 cells were infected, sorted, and cultured as described and RNA was prepared. RNA (10 μg) was analyzed by Northern blot with the full-length p21^WAF1 cDNA. Equal loading is depicted by the 18S ribosomal RNA. (D) Schematic diagram of the 2.3-kb promoter region of the p21^WAF1 gene cloned into the pGL2 reporter vector; ▪, AML1 DNA-binding sites; ▨, putative AML1-binding site (TGTGGG). (E) K562 cells were transfected with 10 μg p21-pGL2, with either 2 μg empty vector or those expressing HA-AML1-ETO, HA-AML1-ETO-R174Q, AML1-ETO, or AML1-ETO-L148D brought to a total of 20μg with 5.6 μg herring sperm DNA, 2 μg of an expression plasmid for CBFβ and 400 ng pRL-null as internal control for transfection, and cultured for 16 to 23 hours. Experiments depict the average and SD of 5 individual experiments with 2 different batches of DNA. (F) ChIP assay for association of AML1 and AML1-ETO to the distal and proximal regions of the p21^WAF1 promoter. Cells were transfected with 10 μg p21pGL2 or Δ1.9p21pGL2 with either 2 μg pCDNA6, pCDNA6-HA-AML1, or pCDNA6-HA-AML1-ETO and following ChIP the isolated DNA fragments were subjected to PCR with specific primers that amplify regions 1 and 2 depicted in panel D, and analyzed by DNA gel electrophoresis.