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Blood, Vol. 112, Issue 13, 5141-5149, December 15, 2008
c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches
Blood Hallaert et al.
112: 5141
Supplemental materials for: Hallaert et al
Files in this Data Supplement:
- Figure S1. RT-MLPA analysis of p53 WT and p53 dysfunctional CLL cells, stimulated via CD40 in absence or presence of kinase inhibitors (JPG, 119 KB)
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Cells were stimulated for 48 hrs as in figure 3b, and apoptotic gene expression profile was analyzed by RT-MLPA. Results were normalized and expressed relative to total signal. Housekeeping genes included in this assay were, GUS, B2M, FLT and Parn. (Note that probes for IAP2 are non functional; Eldering E. et al., NAR., 2003). Graphs represent averaged data ± SD of n=4 p53 functional (A) and n=3 dysfunctional (B) samples.
- Figure S2. Peripheral blood-derived and CD40-stimulated CLL cells are insensitive to imatinib or dasatinib (JPG, 56.9 KB)
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(A–B) Peripheral blood-derived CLL cells (n=3) were stimulated with increasing concentration of imatinib or dasatinib for 24, 48, or 72 hrs. (C–D) CLL cells (n=4) were cultured for 48 hrs in medium or together with 3T3, or 3T40L cells, followed by stimulation with different concentrations imatinib or dasatinib for 48 hrs. The BCR-Abl positive cell line K562 was used as a positive control for imatinib and dasatinib-induced apoptosis. Apoptosis was determined by MitoTracker staining.
- Figure S3. NF-кB inhibitor BAY 11-7082 blocks CD40-mediated Bcl-XL and Bfl-1 upregulation (JPG, 48.1 KB)
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CLL cells were stimulated with or without CD40L for 48 hrs in the presence of ERK inhibitor PD98059 or of BAY11-7082, an irreversible inhibitor of IκB-α phosphorylation (1 and 5 µM). Cells were lysed in SDS-containing buffer and analyzed by westernblot.
- Figure S4. Apoptosis sensitivity of individual CLL samples after co-culture with 3T3 or 3T40L cells, followed by treatment with various drugs in conjunction with ABT-737 (JPG, 31.7 KB)
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CLL samples were cultured as described in legend to Fig. 4, followed by incubation with the indicated drugs in the presence of 1 µM ABT-737. Apoptosis was determined after 24 hrs by Mitotracker staining.
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