Overexpression of CEBPA overrides MN1's inhibitory effects on differentiation and sensitizes MN1 cells to ATRA and vitamin D₃. (A) YFP⁺-sorted U937-mock and MN1 cells were transduced with GFP-expressing CEBPA-retrovirus. YFP⁺/GFP⁺ cells were sorted and used for Western blot analysis to determine CEBPA expression (top panel). Sorted cells were seeded in duplicate at the same density (10⁵/mL) at day 0, and cell numbers were counted during 4 consecutive days of culture. (B) Day 4 cytospin preparations were stained with May-Grünwald-Giemsa (original magnification ×400). (C) CD11b expression was analyzed using FACS. (D) RNA was isolated and subjected to real-time RT-PCR to determine G-CSFR and HPRT expression (mean of triplicates ± SEM). (E) Similarly, YFP⁺ FACS-sorted U937-mock and MN1 cells were transduced with GFP-expressing CEBPA-ER retrovirus, and YFP⁺/GFP⁺ FACS-sorted cells were used for Western blot analysis using anti-CEBPA and GAPDH antibodies. (F) Unsorted cells from panel E were treated for 48 hours with vehicle or the indicated amount of tamoxifen or with the same volume of vehicle (ethanol), and expression of CD11b and CD14 was detected using FACS by gating on GFP⁺/YFP⁺ cells (figure represents one of duplicates). (G-H) Unsorted cells from panel E were treated with 100 nM 4-hydroxytamoxifen (4-HT) in the presence or absence of 1 μM ATRA or 100 nM vitamin D₃, respectively. Expression of CD11b (G, filled bars) and CD14 (H, open bars) was determined using FACS by gating on GFP⁺/YFP⁺ cells. CEBPA-ER indicates mock + CEBPA-ER.