Constitutive and inducible GATA-1 expression in LC precursors. (A) IL-4 promotes CD11b⁺CD1a⁺ cells at the expense of CD207⁺CD1a⁺cells when added at day 5 to LC generation cultures. CD34⁺ cells were induced to differentiate to LCs. Day 5–generated cells were subcultured in LC cytokines in the absence or presence of IL-4 (25 ng/mL) for 4 days. At day 9 cells were harvested and analyzed for CD207 or CD11b versus CD1a. Data are representative of 3 independent experiments. (B-D) Tet-on-inducible system in primary LC precursors. CD34⁺ cells were transduced with retroviral vectors encoding Tet-activator-IRES-lyt2, DOX-inducible GATA-1-IRES-NGFR, or empty vector control. Cells were stimulated with LC-promoting cytokines for 5 days. DOX (2 μg/mL) was added from day 5 to day 7. (B) FACS diagrams show day 5–generated cells (left) or day 7–generated NGFR⁺ cells (right) analyzed for CD207 or CD11b versus CD1a. (C). FACS diagrams represent cells at day 7 analyzed for NGFR expression. (A-C) Each number in a quadrant represents the percentage of cells in the quadrant. (D) Bars represent mean percentages (± SEM; n = 3) of CD1a⁺CD11b⁺ cells from control vector or GATA-1–transduced cells at day 5 (total culture) and day 7 (gated NGFR⁺ cells); *P < .05. (E) LC generation cultures were transduced at day 3 to day 4 with a retroviral vector encoding VDR-IRES-GFP or empty control vector. Forty-eight hours after transduction, GFP⁺ cells were isolated by using FACS. Equal numbers of GFP⁺ cells were used for quantitative real-time RT-PCR analysis. Bars represent VDR mRNA levels in 1 representative experiment (cell numbers in PCR experiments: 3 × 10⁴ to 2 × 10⁵; n = 4).