The BMP2–SMAD–ZNF423 axis in ALL. (A) Intraindividual transcriptome analysis. Heatmap of differentially expressed B lymphoid development–related genes in B precursor ALL versus individually matched normal B progenitor cells (n = 4 matched pairs). ID was given for each gene representative probe set. ZNF423 probe hybridizes to the 3′UTR of the gene. Color code represents Signal Log Ratio (SLR) with ceiling at max −4/+4; * indicates ceiled values. I1s-I3s, initial FACS-sorted leukemia sample; E1-4s, FACS-sorted B progenitor cells at the end of reinduction in complete remission; I4, unsorted initial leukemia sample (>90% blasts). (B–E) qPCR-based quantification of ZNF423 (B), BMP2 (C), SMAD1 (D), and ZNF521 (E) mRNA levels at various stages of hematopoietic development (left) and in primary B cell ALL (n = 200), ESC lines (H1, HES2), and normal hematopoietic cells (right). Values were normalized to β-2-microglobulin, B2M (2^−ΔCt*1000). Error bars represent standard deviation (SD) of technical triplicates. Cutoff is defined as double SD of highest value in the normal compartment. (F) Expression of endogenous ZNF423 protein in primary ALL and ALL cell lines. As a control, 293T cells were transfected with Flag-ZNF423α. ZNF423 was immunoprecipitated (IP) using the ZNF423 monoclonal antibody, IgG2A was used as isotype control. Protein precipitates were immunoblotted for ZNF423 and FLAG-tag. (G) ELISA-based detection of soluble BMP2 in BM, peripheral blood (pBl), and cord blood (cBl) plasma from ALL patients and healthy donors. Lysis buffer and assay diluent display the negative controls. Red horizontal line, threshold value of maximum expression in normal cells. Error bars represent SD of technical triplicates. (H) Immunoblot analysis of SMAD1 and phosphoSMAD1/5 protein expression in primary ALL samples including corresponding SMAD1 transcript levels (refer to D). Actin was used as loading control.