Figure 2.

The effect of CD40 stimulation and Compound A treatment on cIAP levels, NF-κB activity and target gene expression. (a) CLL cells were co-cultured with 3T3 cells (left) or 3T40 cells (right) in the presence of Compound A (250 nM) for 1, 4 and 24 h. Immunoblotting was performed for cIAP1 and cIAP2 on total lysates, and actin was used as a loading control. Blots from a representative CLL patient, of a total of three (left) or 4 (right), are shown. (b) CLL cells co-cultured with 3T40 cells were treated for 4 h with the indicated compounds in the following concentrations: Compound A 250 nM, CHX 20 μg/ml, MG132 500 nM, bafilomycin A 100 nM and ammonium chloride 25 μM. Immunoblotting was performed for cIAP2 on total lysates, and actin was used as a loading control. Blots are representative of two CLL patients. Patient samples used were nos. 39 and 40 from Table 1. (c) CLL cells were co-cultured with 3T3 or 3T40 cells in the presence or absence of Compound A (250 nM) for 24 h. Activity of the NF-κB subunits p65 and p52, as a measure of activity of the canonical and non-canonical NF-κB pathways respectively, was determined using ELISA with the TransAM NFκB family transcription factor assay kit (n=3). Error bars represent S.E.M. (d) CLL cells were treated as in c, but for 48 h. Bcl-X_L and Bfl-1 mRNA expression levels were analyzed by RT-MLPA (n=5), and depicted as percentage of the total signal in that sample. β-Glucuronidase is depicted as a housekeeping gene not responsive to NFκB. Error bars present S.E.M. (e) CLL cells were treated as in c, but for 72 h. Supernatants were collected, and TNFα levels were measured using ELISA (n=16). Error bars represent S.E.M.