Id2 promotes erythropoiesis of erythromyeloid progenitors. (A) Id2 expression levels in EML-GFP and EML-Id2-GFP were determined by Western blot analysis. (B) EML-GFP or EML-Id2-GFP were induced for erythroid development in IMDM containing 10% FBS, 1 × 10−5 M 2-ME, 100 ng/mL mSCF, and 5 U/mL hEPO for 5 days. Erythrocytes were identified by Benzidine staining. (C) EML-GFP or EML-Id2-GFP were seeded at a density of 1 × 104 cells/mL in IMDM with 2.75% methylcellulose, 1 × 10−5 M 2-ME, 30% FBS, 100 ng/mL mSCF, 30 ng/mL mIL-3, and 5 U/mL hEPO. BFU-Es were determined after 10 days. (D) LSKs, CMPs, GMPs, and MEPs were purified from mouse BM according to their surface phenotypes, by multicolor-based sorting techniques. Total RNA was purified from LSKs, CMPs, GMPs, and MEPs, and converted to cDNA. Id2 expression levels in these purified progenitors were determined by real-time PCR. (E) MEP-GFP, MEP-Id2-GFP, CMP-GFP, or CMP-Id2-GFP were induced for erythroid development in IMDM containing 10% FBS, mSCF (100 ng/mL), hTPO (100 ng/mL), and hEPO (40 U/mL) for 4 days, and analyzed for TER119 expression by FACS analysis. (F) EML-GFP and EML-Id2-GFP were induced for myeloid development in IMDM containing 20% HS, mSCF (100 ng/mL), atRA (10 μM), and mIL-3 (30 ng/mL) for 5 days, followed by atRA (10 μM) and mGM-CSF (20 ng/mL) for 5 days, and analyzed for Gr-1 and Mac-1 expression by FACS analysis. CMP-GFP or CMP-Id2-GFP were induced for myeloid development in IMDM containing 10% FBS, mSCF (100 ng/mL), mIL-3 (30 ng/mL), and mGM-CSF (20 ng/mL) for 4 days, and analyzed for Gr-1 and Mac-1 expression by FACS analysis.
