Structure-function analysis of TLX1-mediated transcriptional regulation in ALL-SIL T-ALL cells. (A) TLX1 protein levels inversely correlate with CD55 surface levels. Top, CD55 FACS analysis. Histogram colors indicate: blue, TLX1Low; green, TLX1Med; and red, TLX1High. Bottom, TLX1 Western blot. TLX1High levels were 6.5× higher and TLX1Med levels were 4.9× higher than TLX1Low levels (P < 0.0001). The difference between the TLX1High and TLX1Med levels was statistically significant (P < 0.05). (B) Venn diagram comparing candidate TLX1 target genes derived from two different microarray technologies: Affymetrix data have been described ; cDNA array targets were selected based on correlation of expression levels with TLX1 protein levels (r > 0.9 or < -0.9; 1% FDR; n = 3-4). (C) Hierarchical clustering of genes identified in both profiling experiments. (D) TLX1 levels in knockdown and "rescued" ALL-SIL populations: top, Western blot; bottom qRT-PCR. The TLX1 knockdown populations expressing coding regions of wild-type or mutant forms of TLX1 are: TLX1 WT, wild-type; TLX1 N51A, DNA binding-deficient mutant; TLX1 F19E, TLE binding-deficient mutant; shTLX1 GFP, TLX1 knockdown plus GFP reporter. Total or endogenous TLX1 mRNA levels were determined using primers targeting coding or 3' noncoding regions of TLX1, respectively. The data is normalized to TLX1 knockdown expressing GFP alone (shTLX1 GFP). (E) TLX1 signature gene expression in the populations described in D. The data is normalized to TLX1 knockdown expressing GFP alone (P values are indicated in the text and Additional file 4; n = 2-3 biological replicates, each comprising 3 technical replicates).