ZAP-70 constitutively regulates gene expression relevant for T-cell interactions. (A) PCA, created for the top 2000 most abundant genes, illustrates the separation across patients (mainly captured in PC1) and treatments (PC2). (B) Venn heat map representing the Jaccard Similarity Index of the top 2000 most abundant genes, across all pairwise comparisons on patients and treatments. The Jaccard Similarity Index spans the 0 to 1 range, which corresponds to no overlap (0) to identical sets of genes (1). The clear separation between patients is contrasted by closer similarity on replicates and treatments. (C) Volcano plot illustrating the differentially expressed genes [the x-axis is the log2(FC) between the control and siZAP-70 samples; the y-axis is the −log10(P value)]. Gray points correspond to nondifferentially expressed genes, red points correspond to genes for which the P value is <.05 [but |log2(FC)| < 0.5], blue points are significantly differentially expressed genes with log2(FC) > 0.5, and color intensity is proportional to the log2 of the average normalized expression levels across the compared samples. (D) Heat map of genes from cells of 2 individual primary patients with CLL treated with NSC siRNA or ZAP-70 siRNA. Red and blue indicate relatively high and low expression, respectively. Each condition analyzed depicts technical replicates, derived from 2 individual siRNA transfections. Genes were ranked by average Log2(FC), using EdgeR analysis, from bottom to top. (E) qRT-PCR analysis of CD1c and IL4I1 in primary CLL cells (CD19⁺ selected) (n = 8 and n = 7, respectively) transfected with nonspecific siRNA or ZAP-70 siRNA and monocultured for 24 hours. Relative gene expression level is normalized to reference gene GAPDH and compared with NSC. (F) qRT-PCR analysis of CCL3 and CCL4 in primary CLL cells (CD19⁺ selected) (n = 8 and n = 7, respectively) transfected with nonspecific siRNA or ZAP-70 siRNA and monocultured for 24 hours. Relative gene expression level is normalized to reference gene GAPDH and compared with NSC. (G) qRT-PCR analysis of CCL3 and CCL4 in primary CLL cells (CD19⁺ selected) (n = 4) transfected with nonspecific siRNA or ZAP-70 siRNA. Cells were treated with ibrutinib at 0.5 μM for 24 hours before cell harvesting. Relative gene expression level is normalized to reference gene GAPDH and compared with NSC. (H) GO enrichment analysis of the differentially expressed genes in (C). Background genes are the set of all genes that pass the noise threshold. *P < .05, **P < .01. ns, not significant (P > .05).