BASH and active forms of PKCη and Raf-1 induce κ gene rearrangement in synergy with PMA treatment in the pre-B-cell line. (A) BKO84 cells were infected with the retroviruses carrying pMX-IRES-rCD2 (rCD2), pMX-BASH-IRES-rCD2 (BASH/rCD2), pMX-PKCη-IRES-rCD2 (PKCη/rCD2), or pMX-Raf-1-IRES-rCD2 (Raf-1/rCD2). Four days after the infection, rCD2⁺ cells were sorted by MACS. Genomic DNAs from the sorted samples, from BKO84 cells treated with PMA for 2 days (PMA) or not (-), and from mouse tail (Tail) or ES cells (ES) were subjected to PCR-detecting κ gene rearrangements (top) and LM-PCR-detecting Jκ-associated DNA breaks (middle) followed by Southern blot analysis. The amount of genomic DNA used for these assays was monitored as in Figure 2D (bottom). (B) BKO84 cells were infected as in panel A. Four days after the infection, rCD2⁺ (left) or rCD2^- (middle) cells were separated by MACS, fixed and permeabilized, stained with PI, and analyzed for DNA contents by flow cytometry. The percentages of the cells in sub-G₁ (apoptotic), G₁, and S/G₂/M (percentages of nonapoptotic cells in parentheses) are denoted in each histogram. FSC profiles of the rCD2⁺ (thick line) and the rCD2^- (dotted line) cells within a live lymphocyte gate (defined by FSC/side scatter [SSC] profile) are shown as histograms (right). (C) BKO84 cells were infected with the indicated retroviruses, stimulated with PMA (PMA) or solvent alone (-) from day 3 after infection, and analyzed for κ expression by flow cytometry on day 5. The bar graph indicates the frequency of κ-positive cells among GFP-positive (+) or GFP-negative (-) cells. (D) Pre-BCR signaling pathway regulating the κ gene rearrangement. A hypothetical “docking area” is indicated as a gray part. DAG indicates diacylglycerol; X, a hypothetical scaffold protein for the BASH-nPKC-Raf-1 pathway. See “Discussion” for more details.