Knock-down of Id2 by shRNA induces B-cell development. (A) Illustration of pRetro-NS-shRNA and pRetro-Id2-shRNA retroviral vector (left panel). Id2 expression levels in BMC-NS-shRNA and BMC-Id2-shRNA were determined by Western blot analysis (middle panel). Id2 expression levels in EML-NS-shRNA and EML-Id2-shRNA were determined by Western blot analysis (right panel). (B) BMC-NS-shRNA and BMC-Id2-shRNA were transplanted into lethally irradiated mice with supporting BMCs. A representative from 3 independent experiments is shown here. Eight mice were analyzed in each transplantation. Hematopoietic reconstitution in recipient BM was examined 4 weeks after transplantation by analyzing the percentage and the total cellularity of GFP-positive cells (top panels). B-cell reconstitution was examined 4 weeks after transplantation in BM by FACS analysis using B220 and CD43 antibodies (bottom panels). (C) Hematopoietic reconstitution in recipient spleen was examined 4 weeks after transplantation by analyzing the percentage and the total cellularity of GFP-positive cells (top panels). B-cell reconstitution was examined 4 weeks after transplantation in spleen by FACS analysis using IgM and IgD antibodies (bottom panels). (D) FACS analysis of B cell–specific cell surface marker B220 in EML-NS-shRNA and EML-Id2-shRNA. A representative from 3 independent experiments is shown here. (E) Real-time PCR analysis of B cell–specific gene expression in EML-NS-shRNA and EML-Id2-shRNA. A representative from 3 independent experiments is shown here.