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Blood, Vol. 112, Issue 4, 1413-1423, August 15, 2008
Up-regulation of WRN and DNA ligase III in chronic myeloid leukemia: consequences for the repair of DNA double-strand breaks
Blood Sallmyr et al.
112: 1413
Supplemental materials for: Sallmyr et al
Files in this Data Supplement:
- Table S1. Patient samples (PDF, 20.1 KB)
- Table S2. γ-H2AX foci formation in siRNA treated K562 with and without IR (PDF, 24.5 KB)
- Table S3. γ-H2AX foci formation in siRNA treated NC10 with and without IR (PDF, 24.5 KB)
- Table S4. In vivo plasmid end-joining assay in siRNA-treated K562 (PDF, 32.5 KB)
- Table S5. In vivo plasmid end-joining assay in siRNA-treated NC10 (PDF, 32.5 KB)
- Figure S1. K562 shows higher level of ROS compared to NC10 (JPG, 44.1 KB)
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Intracellular ROS level were determined by using the probe 2,7-dichlorodihydro-fluorescein diacetate (H2DCF-DA, Invitrogen, Carlsbad, CA). Cells (1 × 106) were treated in the presence of the probe to a final concentration of 10 µM and incubated at 37°C for 10 minutes. ROS levels were determined by flow cytometry (Becton Dickinson FACScan).
- Figure S2. Western blot of MO7e and P210MO7e showing the expression of Artemis and DNA ligase IV (JPG, 52.1 KB)
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Western blot analysis of Artemis and DNA ligase IV using nuclear extracts from BCR-ABL–negative cell line (MO7e) and BCR-ABL–positive cell line (P210MO7e). Actin was used as loading control.
- Figure S3. Co-localization of DRneo (JPG, 40.1 KB)
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(A) Images of cells showing co-localization of DRneo (FITC, green signal) and γH2AX, Ku86, DNA ligase IV, DNA ligase IIIα and WRN (TRITC, red signal) in NC10DRneo cells transfected with ISce1. Nuclei are stained with DAPI (blue). Right panels are merged images of FITC and TRITC showing co-localization. (B) Histogram showing the percentage of co-localization in both K562DRneo (black bar) and NC10DRneo cells (gray bar).
- Figure S4. Western blot of siRNA treated K562 and NC10 (JPG, 51.6 KB)
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K562 and NC10 cells were siRNA treated with DNA ligase IIIα and WRN. Nuclear extracts were prepared 72 hours after transfection. Western blotting was performed using DNA ligase IIIα (75% knockdown in K562 and NC10; upper panel) and WRN antibody (90% knockdown in K562 and 75% knockdown in NC10; middle panel). siRNA using non-target oligonucleotides were used as control. Actin was used as loading control (lower panel).
- Figure S5. Viability (%) measured using trypan blue dye exclusion (JPG, 26.8 KB)
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(A) Table of % viability in siRNA Nontarget, siRNA WRN and siRNA DNA ligase IIIα experiment in K562 cells. (B) Histogram showing the percentage of cell viability. Error bars reflect the standard error of the mean.
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