TRAF4 is upregulated in CXCR4^dimCD5^bright intraclonal subpopulation. (A) Heatmap of differentially expressed mRNAs (fold-change > 2; adjusted P < .0005) in 10 pairs of CXCR4/CD5 sorted subpopulations (purity > 99%; for sample characteristics, see supplemental Table 1). Plotted mRNAs represent an overlap of differentially expressed mRNAs and predicted evolutionary conserved miR-29 targets (TargetScan tool). Samples CLL1, CLL4, CLL6, CLL8, CLL9, and CLL14 are identical to Figure 1A (6 of 7 pairs for miRNA profiling). Heatmap was generated from counts per million reads (rows centered to the median of the row). For details on mRNA expression and miR-29 target prediction see supplemental Table 4. (B) TRAF4 mRNA levels analyzed using qRT-PCR in the CXCR4/CD5 sorted subpopulations from 10 primary CLL samples. Differences were compared by Wilcoxon matched pairs test. (C) Intracellular staining for TRAF4 protein levels in the CXCR4/CD5 subpopulations from 13 CLL samples. Results are presented as the ratio of TRAF4 expression to the isotype control, and the statistical differences were compared by paired t test. (D) The expression of TRAF4 protein in CXCR4^dimCD5^bright, CXCR4^{intermediate(int)}CD5^{intermediate(int)}, and CXCR4^brightCD5^dim intraclonal cell populations. (i) Representative immunoblot blot analysis of TRAF4 in sorted CXCR4/CD5 subpopulations from 2 patient samples. Histon H3 was used as a loading control. (ii) Statistical analysis of TRAF4 protein levels in the CXCR4/CD5 sorted subpopulations (n = 5). The statistical differences were tested by paired t test, and the error bars indicate the standard error of the mean (SEM). (iii) Representative example of a gating strategy for CXCR4^dimCD5^bright, CXCR4^{intermediate(int)}CD5^{intermediate(int)}, and CXCR4^brightCD5^dim intraclonal cell populations. The CXCR4^intCD5^int represents a transitional subpopulation between CXCR4^dimCD5^bright and CXCR4^brightCD5^dim cells.