BAFF-mediated survival is blocked by VAY-736 treatment of CLL B cells. (A) Representative annexin V/propidium iodide (PI) flow cytometry analysis of a CLL patient B-cell viability at 72 hours. (B) Time course comparison of primary CLL cells’ viability at 24, 48, and 72 hours treated with BAFF (500 ng/mL), VAY-736, or cells pretreated with VAY-736 subsequently stimulated with BAFF (P < .001: 72 hours, BAFF vs untreated; P < .01: 48 hours, VAY-736 + BAFF vs BAFF). Inhibitory effect of VAY-736 at each time point was tested by interaction contrast (BAFF + VAY-736 subtracting BAFF vs VAY-736 subtracting untreated; 72 hours, P < .01). Data were analyzed by using a mixed effect model, and Holm’s method was used to adjust multiplicity (n = 21 patients). (C-E) Western blot analysis of p100 and p52 protein levels in primary CLL patient B cells. Treatments were soluble BAFF (500 ng/mL) for 16 hours with or without pretreatment with VAY-736 (10 μg/mL). Activation of alternative NF-κB was determined by separate cytoplasmic (CE) and nuclear (NE) protein fractions, loading controls actin (cytoplasmic), and Lamin B (nuclear). Quantification of p100 or p52 protein by densitometry analysis was normalized to loading control and then to the untreated condition (P < .01: cytoplasmic p100, VAY-736 + BAFF vs BAFF; P < .05: p52 cytoplasmic and nuclear levels, BAFF vs untreated, VAY-736 + BAFF vs BAFF; n = 5 CLL patients, 3 independent experiments). *P < .05, **P < .01, and ***P < .001.