CEBPA overrides the MN1-induced inhibition of differentiation of primary mouse lin⁻BM cells. (A) The protocol used for generation of mock (YFP⁺/GFP⁻), MN1 (YFP⁺/GFP⁻), mock + CEBA-ER (YFP⁺/GFP⁺), and MN1 + CEBPA-ER (YFP⁺/GFP⁺) transduced primary mouse BM cell populations. (B) Aliquots of cells were analyzed using FACS after a second lineage depletion at the day 0 time point (before 4-HT treatment). Expression of Sca1 and c-Kit surface markers was determined of untreated YFP⁺ (top panels: mock; bottom panels: MN1) or YFP⁺/GFP⁺ (top panels: mock + CEBPA-ER; bottom panels: MN1 + CEBPA-ER) populations. (C) Similarly, Mac1/Gr1 expression was analyzed using the indicated gates at day 0 (before 4-HT treatment). The percentage of Mac1⁻/Gr1⁺(1) or Mac1⁺/Gr1⁺(2) granulocytes and Mac1⁺/Gr1⁻(3) monocytes is shown in mock and MN1 cells (without CEBPA-ER), and in mock + CEBPA-ER, MN1 + CEBPA-ER, or CEBPA-ER populations. (D) Day 0 cells were seeded (2 × 10⁵/mL) in duplicate with vehicle or 4-HT (100 nM) in the presence of SCF, IL-3, and IL-6. Two days later, Mac1/Gr1 expression was determined using FACS of YFP⁺/GFP⁻ gated cells in mock and MN1 cells treated with vehicle [4-HT (−)] or 4-HT (mean of duplicates ± SEM). *P < .04; **P = .007; ***P < .001. (E) Similarly, Mac1/Gr1 expression was detected at day 2 in mock + CEBPA-ER (top panels) and MN1 + CEBPA-ER cells (bottom panels), treated with vehicle or 4-HT. Figure represents one of the duplicates. (F) In an independent experiment, without pre-5-fluorouracil treatment of the mice, lineage-depleted primary mouse BM cells were cotransduced with GFP-expressing mock or MN1- and YFP-expressing CEBPA retroviruses. Four days after transduction, 300 or 500 cells were FACS-sorted (mock or MN1, GFP⁺/YFP⁻; CEBPA, GFP⁻/YFP⁺; MN1 + CEBPA, YFP⁺/GFP⁺) and plated in methylcellulose media in duplicate. Colonies in each dish were counted 8 days later. Graph represents mean of duplicates ± SEM. represents 100 cells/dish; , 166 cells/dish.