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Blood, Vol. 105, Issue 12, 4776-4783, June 15, 2005
Human chronic lymphocytic leukemia B cells can escape DNA damage-induced apoptosis through the nonhomologous end-joining DNA repair pathway
Blood Deriano et al.
105: 4776
Supplemental materials for: Deriano et al, Vol 105, Issue 12, 4776-4784
Files in this Data Supplement:
- Figure S1. NHEJ DNA repair pathway is over activated in resistant B-CLL cells after DNA damage (JPG, 36 KB)
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Supplement to Figure 3. (A) NHEJ activity in untreated cells (indicated by – in the Irradiation row) and 15 minutes after receiving 10 Gy irradiation (indicated by + in the Irradiation row). Assays were carried out using 3 resistant (R1, R2, and R3) and 3 sensitive B-CLL cell samples (S1, S2, and S3). (B) Changes in NHEJ activity after 10 Gy irradiation for each B-CLL sample.
- Figure S2. Early increase in DNA-PKcs activity in resistant B-CLL cell extracts after γ-irradiation (JPG, 27 KB)
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Supplement to Figure 4. DNA-PKcs activity in additional pairs of resistant (R2 and R3) and sensitive (S2 and S3) B-CLL cell nuclear protein extracts (25 µg/lane) at the indicated times after irradiation (10 Gy).
- Figure S3. Constitutive increase in Ku70/80 heterodimer DNA-end binding activity (JPG, 23 KB)
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Supplement to Figure 5. Ku DNA-end binding activity in whole protein extracts from 3 resistant (R1, R2, and R3) and 3 sensitive (S1, S2, and S3) B-CLL samples (1 µg/lane) at the indicated times after irradiation (10G y).
- Figure S4. B-CLL cells are arrested in G0/G1 phase of the cell cycle (JPG, 61 KB)
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Cell-cycle fractions were determined by flow cytometric quantification of DNA content using propidium iodide (PI) labeling. After ethanol fixation (70% v/v) and RNase (30 µg/mL) treatment, cellular DNA was stained with PI (50 µg/mL). PI-DNA fluorescence was acquired on a FACSort (Becton Dickinson Immunocytometry System, San Jose, CA) using linear amplification into FL2 channel (585/20BP), and cell-cycle fractions were calculated using Modfit 3.2 software (Verity Software, Topsham, ME). Over 90% of both resistant (R1, R2, and R3) and sensitive (S1, S2, and S3) B-CLL cells were in G0/G1 phase. In theory, a small proportion of cells (<5%) may have been G2, but since the proportions of S-phase cells were lower than 1%, we assumed that the most of this peak was doublets due to the ethanol fixation. Under the same experimental conditions, cultured cell lines (a B-cell line, MO59K and MO59J), showed cell-cycle fractions typical of cycling populations: lower G1 fractions (B-cell line, 75%; MO59K, 60%; MO59J, 50% for the diploid population and 60% for the aneuploid population), high S phase (B-cell line, 22%; MO59K, 25%; MO59J, 33% for the diploid population and 25% for the aneuploid population), and high G2/M fractions (B-cell line, 3%; MO59K, 15%; MO59J, 16% for the diploid population and 15% for the aneuploid population).
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