C/EBPα-C^m had the strong ability to block myeloid differentiation. (A) Schematic diagram of C/EBPα-WT (p42 and p30) and mutants, T60fsX159 (C/EBPα-N^m) and 304_323 dup (C/EBPα-C^m). TAD indicates the transcriptional activation domain; bZIP, basic region leucine zipper domain. (B) Expression of C/EBPα-WT and its mutants in Plat-E cells transiently transfected with a Myc-tagged C/EBPα-WT, C/EBPα-C^m, C/EBPα-N^m, or C/EBPα-p30 or an empty vector (pMXs-IP) and expression of C/EBPα mutants in 32Dcl3 cells transduced with Myc-tagged C/EBPα-C^m, C/EBPα-N^m, C/EBPα-p30, or mock (pMXs-IP). Cell lysates were subject to immunoblotting with anti-Myc Ab or anti-tubulin Ab as control. The results shown are representative of 3 independent experiments. (C) The growth of 32Dcl3 cells transduced with Myc-tagged C/EBPα-C^m, C/EBPα-N^m, C/EBPα-p30, or mock (pMXs-IP) in the presence of 1 ng/mL IL-3. All data points correspond to the mean and the standard deviation (SD) of 3 independent experiments. (D-E) 32Dcl3 cells transduced with Myc-tagged C/EBPα-C^m, C/EBPα-N^m, C/EBPα-p30, or mock (pMXs-IP) were cultured in the presence of 50 ng/mL G-CSF for 6 days. (D) Morphology of these cells was assessed by Giemsa staining. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); objective lens, UplanFl (Olympus); original magnification ×40. (E) Surface expression of CD11b in these transfectants after incubation with 1 ng/mL IL-3 (bold histograms) or 50 ng/mL G-CSF (filled histograms) for 6 days was analyzed by flow cytometry. The result of control staining is shown as a thin-lined histogram. Data are representative of 3 independent experiments. (F) Relative expression levels of G-CSF-R in 32Dcl3 cells transduced with Myc-tagged C/EBPα-C^m, C/EBPα-N^m, C/EBPα-p30, or mock (pMXs-IP) were estimated by using real-time PCR. Data are representative of 3 independent experiments.