Increased frequency of a premitotic state among HTLV-transformed cells with high 4-1BB expression. (A) Expression of 4-1BB and ligand was determined in the ATL-derived cell line Champ. Extracellular 4-1BB and 4-BBL were stained with specific antibodies (black curves) and analyzed by flow cytometry; gray curves represent an unspecific isotype-matched control antibody. Intracellular PI staining is shown (black curves) in comparison to an unstained control (gray, dashed). One representative experiment of at least 3 is shown. (B) Simultaneous analysis of 4-1BB expression on the cell surface and staining of DNA-content by PI is shown in a representative dot plot of the ATL cell line Champ. Gates were set for cells in G1/G0, S, and G2/M phases. (C) Proportion of cells in the S and G2/M phase (premitotic cells) were calculated for 4-1BBhigh and 4-1BBlow cells of cell lines Eva, StEd, HuT-102, and Champ. The means of 3 independent experiments plus or minus standard error are shown.