Ibrutinib affects pre-BCR signaling pathway components and cell migration. (A) Results of gene expression profiling for selected genes of pre-BCR⁺ B-ALL cells (RCH-ACV) treated with ibrutinib for 24 hours (left bar in each pair) or 72 hours (right bar in each pair). Values were normalized to controls (DMSO-treated cells); data from multiple probes were averaged when available; displayed are the mean ± SD. (B) PTPN6 and BCL6 protein levels were reduced in pre-BCR⁺ (RCH-ACV, SMS-SB, Kasumi-2), but not in pre-BCR⁻ (NALM-21, REH), B-ALL cells after treatment with 0.5 µM of ibrutinib for 72 hours. BCL6 and PTPN6 signals were quantified by densitometry and normalized to corresponding GAPDH measurements and subsequently to untreated controls. Short and long exp., short and long exposition. (C-E) CD72, CD22, and CD44 surface expression after 72 hours of ibrutinib treatment as measured by flow cytometry. Results of 3–5 independent experiments presented as the mean fluorescence intensity ratio, mean with 95% CI. (F) Pretreatment with ibrutinib inhibited migration of ALL cells toward CXCL12 in chamber chemotaxis assay. Numbers of transmigrated cells were measured in triplicate in 3–4 independent experiments, were normalized to 1 : 20 dilution of input cells, and are presented as the mean with 95% CI. (G) Treatment with ibrutinib for 72 hours significantly decreased spontaneous migration of RCH-ACV cells beneath KUSA-H1 stromal cells. Results of 2 independent experiments are displayed as the mean with 95% CI. MFI, mean fluorescence intensity. ns, not significant. *P < .05; **P < .01; ***P < .001.