Sox4 induces CK1ε expression and negatively regulates Wnt/β-catenin signaling. (A) Real-time RT-PCR analysis of Sox4 and CK1ε expression in pro-B cells sorted from the bone marrow of Sox4^fl/+Vav-Cre and Sox4^fl/flVav-Cre mice (left), fraction B Sox4^fl/+ and Sox4^fl/fl pro-B cells sorted from cultures of 9 days post-SE-Cre-transduction (middle), and HA- or HA-Sox4-expressing p190 Bcr-Abl-immortalized pro-B cells that had the endogenous floxed Sox4 deletion (right). In each pair, the higher expression was set as 1. (B) Genome browser view of Sox4 binding in the CK1ε locus identified by ChIP-Seq. CK1ε transcript in relation to Sox4 binding is shown. The arrow points to the identified Sox4-binding site. (C) qPCR detection of the Sox4 binding to CK1ε locus. Sox4 binding to proximal promoter of CK1ε locus was detected by qPCR amplification of bioChIP DNA from BAP or BAP-Sox4 cells, and the results were normalized to those from corresponding input DNA samples (normalized results from BAP cells were set as 1). (D) Real-time RT-PCR analysis of CK1ε expression in pro-B cells transduced with NT-shRNA or 2 different shRNAs specific for CK1ε. Expression in nontransduced cells was set as 1. (E) Effect of CK1ε silencing in pro-B cells on the transition from fraction B to fraction C. Fraction B cells were sorted out after 3 weeks of NT or CK1ε shRNA transduction and cultured for an additional 6 days before flow cytometry analysis. Numbers indicate percentages of cells in each quadrant. (F) Immunoblot analysis of the levels of β-catenin, p-GSK3β, and GSK3β in total cell lysates from NT or CK1ε shRNA-expressing pro-B cells. α-Tubulin served as a loading control. The histogram shows the relative band intensity normalized to that of α-tubulin. (G) Schematic representation of dual reporter vectors. PGK promoter drives the expression of mCherry in the forward orientation, and Sox4 or β-catenin interacting Tcf elements drive luciferase reporter expression in the opposite orientation. LTR, long terminal repeat; RE, response element; SIN, self-inactivating (see supplemental Methods for details of vector construction). (H) Effect of CK1ε silencing on the Wnt/β-catenin luciferase reporter activity in pro-B cells. (I) Sox4 and Wnt/β-catenin luciferase reporter activity in pro-B cells with or without Sox4 deletion. Data are representative of 1 (B-C), 2 (F), or 3 (A,D-E,H-I) independent experiments; error bars (A,C-D,H-I), SEM.