Secretion of soluble factors is not involved in the impaired proliferation of TF-1-fms-Nef-ER cells induced by Nef activation. Parental TF-1-fms cells, but not TF-1-fms-Nef-ER cells, were engineered to express EGFP protein by the retroviral infection system. The cell density of the TF-1-fms-EGFP (parent-EGFP) and TF-1-fms-Nef-ER (Nef-ER) cells was adjusted to 1 × 10⁴ cells/mL, and the cells were cultured after an equal volume of the cell suspensions was combined. These cultures were incubated for 3 days with M-CSF in the presence or the absence of 4-HT. (A) After the cocultures, cells were subjected to flow cytometric analysis to determine the relative cell numbers. (B) Absolute cell numbers of the parental cells and Nef-ER cells were calculated from the relative cell numbers (A) and the total cell numbers in the cocultures. Error bars from triplicate assays are shown and represent SD. These results are representative of 3 independent experiments.