Identification and characterization of the Sox4 transcriptional network. (A) Schematic representation of HA-Sox4 complementation experiments that required sequential transduction of 3 viral vectors. After each step, we sorted out cells positive for appropriate markers by flow cytometry. First, cultured fetal liver pro-B cells from Sox4^fl/fl;Rosa26eYFP mouse embryos were immortalized by p190 Bcr-Abl (with mCherry as a marker). Second, SE-Cre was introduced, resulting in a deletion of floxed Sox4 and the STOP cassette before the eYFP sequence, and the cells became eYFP⁺. Finally, HA-Sox4 or HA (with mCerulean as a marker) was introduced. The reason we used p190 Bcr-Abl to immortalize the pro-B cells was that ectopic expression of Sox4 in primary pro-B cells instantaneously impeded cell survival, and immortalization of pro-B cells with p190 Bcr-Abl could overcome this problem. Moreover, this sequential introduction of vectors minimized potential confounding effects of BCR-ABL as the HA-Sox4-expressing experimental cells and HA-expressing control cells were generated from the same group of BCR-ABL⁺ cells. (B) Immunoblot analysis of Sox4 expression in HA-Sox4- or HA-transduced and immortalized pro-B cells that were depleted of endogenous Sox4. α-Tubulin served as a loading control. (C) Transcription activation by HA-Sox4 in luciferase reporter assays of 293T cells (error bars, standard error of the mean [SEM]). (D) Effect of HA-Sox4 expression on the differentiation from fraction B to fraction C of immortalized pro-B cells with a deletion of endogenous Sox4 (Sox4^fl/flSE-Cre). Cells were analyzed by flow cytometry 7 days after introduction of HA or HA-Sox4. Numbers indicate percentages of cells in each quadrant. (E) Heat map of gene expression microarray profiling of cultured pro-B cells for loss of function (left, Sox4^fl/fl vs Sox4^fl/flSE-Cre) and gain of function (right, Sox4^fl/flSE-Cre HA vs Sox4^fl/flSE-Cre HA-Sox4). Expression of the top 100 differentially expressed genes (log2 fold change exceeding ±0.75 and P < .01) is depicted. Scale bar, intensity of signal. Each column represents an independent sample, and each row represents a gene. (F) Overlap between gene expression microarrays for loss (blue oval) and gain (yellow oval) of function of Sox4. Black and red numbers indicate down- and upregulated genes, respectively, in the loss-of-function array and vice versa in the gain-of-function array. (G-H) Ingenuity pathway analysis of gene expression profiles from the Sox4 gain-of-function array. Genes that showed a log2 fold change exceeding ±1.5 were analyzed. The functions of the differentially expressed genes in development (G) and canonical signaling pathways (H) are shown. Data are representative of 2 (B-C), 3 (D), or 1 (E-H) experiment.