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Blood, Vol. 110, Issue 2, 651-660, July 15, 2007
Enhanced phosphorylation of Nbs1, a member of DNA repair/checkpoint complex Mre11-RAD50-Nbs1, can be targeted to increase the efficacy of imatinib mesylate against BCR/ABL-positive leukemia cells
Blood Rink et al.
110: 651
Supplemental materials for Rink et al, Vol 110, Issue 2, 651-660
Files in this Data Supplement:
- Figure S1. Enhanced activation of the ATM kinase in BCR/ABL-positive leukemia cells (JPG, 43.8 KB)
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BCR/ABL (B/A)-positive or parental (P) M07e cells were untreated or treated with MMC for 8 hours. (Upper panel) Activation of ATM was assessed in total cell lysates by SDS-PAGE followed by Western blotting using antibodies recognizing ATM phosphorylated on S1981 (upper box) and total ATM (lower box). DNA damage-dependent autophosphorylation of ATM on S1981 initiates its catalytic activity.1 (Lower panel) ATM kinase assay was performed according to Adamson et al.2 Briefly, ATM immunoprecipitates obtained from the indicated cells were used for the kinase reaction with the GST-p53 as a substrate. The reactions were resolved by SDS-PAGE and examined by Western blotting using antibodies recognizing p53 phosphoserine-15 (upper box) and GST (lower box).
- Figure S2. Time-dependent enhancement of pNbs1 in CML-BC cells (JPG, 50 KB)
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CD34+ cells were isolated from healthy donors (Normal) and CML-blast crisis (CML-BC) patients. Cells were treated with 0.5 µg/mL MMC for 0, 4, 8, and 12 hours in the absence of growth factors. pNbs1, Nbs1, Mre11, and Rad50 were detected by Western analysis.
- Figure S3. Downregulation of Nbs1 in BCR/ABL-M07e cells decreased resistance to MMC (JPG, 52.8 KB)
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M07e (P) and BCR/ABL-M07e (B/A) cells were transfected with siRNA specific for Nbs1 (siS) or nontargeting control siRNA, (siC). Ninety-six hours after electroporation, cells were plated in methylcellulose in the presence of GM-CSF and the indicated doses of MMC. Nbs1 and tubulin expression was examined via Western analysis to confirm Nbs1 downregulation. Colonies were counted after 7 days. Results represent the percent of colonies (± SD) in comparison to untreated group; P<0.01.
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