TF-1- fms cells expressing Nef-ER show impaired proliferation and apoptotic cell death on Nef activation. (A) Schematic diagram of the Nef-ER-IRES-puro construct. ER indicates estrogen receptor hormone-binding domain; IRES, internal ribosomal entry sequence; puro, puromycin resistance gene. (B) Total cell lysates from the parental TF-1-fms cells (parent) or the TF-1-fms clone stably expressing Nef-ER (Nef-ER) were analyzed for the expression of Nef-ER by Western blotting with -Nef rabbit antiserum. (C) Parental TF-1-fms (□) or TF-1-fms-Nef-ER cells (▦) were seeded at a density of 1 × 10⁴ cells/mL in the presence of M-CSF (100 ng/mL) and increasing concentrations of 4-HT. Cells were cultured for 2 days (top row) or 4 days (bottom row), and viable cells were enumerated. Error bars from triplicate assays are shown. Results are representative of 3 independent experiments. Error bars indicate standard deviation (SD). (D) TF-1-fms-Nef-ER cells were cultured with M-CSF in the absence (top) or the presence of 1 μM 4-HT (bottom) for 48 hours. Apoptotic subdiploid cells was detected by flow cytometry. The percentages of subdiploid cells are shown. (E) Parental TF-1-fms (top row) or TF-1-fms-Nef-ER cells (bottom row) were cultured with M-CSF in the absence (left column) or the presence of 1 μM 4-HT (right column) for 24 hours. Cells were analyzed for the presence of apoptotic cells by staining with annexin V-PE, and 7-AAD (lower right quadrant). The percentages in cells of the upper right corners and lower right corners are shown.