Accumulation of GAB1 in CXCR4^brightCD5^dim intraclonal CLL cell subpopulation. (A) Competitive migration of CXCR4/CD5 CLL cell subpopulation into the spleen of NSG mice and their relative retention in the blood (migration continued for 4 hours, n = 6). Cells were sorted according to FCS-A and CD5 (FCS^dimCD5^dim correspond to CXCR4^brightCD5^dim cells and FCS^brightCD5^bright correspond to CXCR4^dimCD5^bright cells; for cell-sorting strategy, see supplemental Figure 1). CXCR4 labeling was not used for cell sorting because it would block CXCR4 interaction with SDF1. One blood sample was excluded from the analysis for technical reasons. (B) Representative gating strategy for CLL cells sorted according to CXCR4/CD5 expression (each subpopulation gated as ∼5% of cells). This sorting, directly based on CXCR4/CD5 labeling, was used for RNAseq profiling and all experiments other than migration assays. (C) Heat map of differentially expressed genes in CXCR4/CD5 subpopulations (fold change, >1.5; P < .001; n = 10), subsequently overlapped with a database of migration-related genes (GO:0016477).⁴⁷  Lower expression is indicated in blue, higher expression in yellow. Gene names marked in bold were validated by real-time qPCR (see Figure 1D and supplemental Figure 5). Lists of top differentially expressed genes, differentially expressed migration-related genes, and Gene Ontology Term analysis are provided in supplemental Tables 2-4. For the volcano plot, sample correlation, and principal component analysis, see supplemental Figures 3 and 4. For patient characteristics, see supplemental Table 1. (D) Validation of GAB1 mRNA expression (real-time qPCR) in primary CLL samples sorted according to CXCR4/CD5 expression (n = 8). (E) Intracellular flow cytometry staining for GAB1 protein in CXCR4/CD5 subpopulations (n = 22). Normalized mean fluorescence intensity (MFI) was calculated as the ratio between the sample and control sample stained only with a secondary antibody. (F) Representative immunoblot from primary CLL samples sorted according to CXCR4/CD5 expression (purity >99.9%). (G) Densitometric quantification of relative GAB1 protein level analyzed by immunoblot (for individual immunoblots, see Figure 3G and supplemental Figure 6A; for densitometric quantification of other proteins, see supplemental Figure 6B-C).