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Blood, Vol. 110, Issue 8, 2996-3004, October 15, 2007
The Myc-evoked DNA damage response accounts for treatment resistance in primary lymphomas in vivo
Blood Reimann et al.
110: 2996
Supplemental materials for Reimann et al, Vol 110, Issue 8, 2996-3004.
Files in this Data Supplement:
- Figure S1. Myc signaling provokes a PP5/Atm-dependent p53 response in vitro (JPG, 125 KB).
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(A) Expression of Myc, Atm, PP5, and Tubulin as a loading control by immunoblot analysis of lysates from NIH3T3 cells and MycERTAM-3T3 (ARF-deficient NIH3T3 fibroblasts stably expressing the inducible c-Myc/estrogen receptor fusion protein that readily translocates to the nucleus upon binding to 4-OH-tamoxifen OHT) stably expressing siRNA against Atm, protein phosphatase 5 (PP5), or mock infection (−). (B) Immunoblot analysis of p53, p53-P-S18 and Tubulin as a loading control 24 hours after exposure to ADR in MycERTAM-3T3 cells stably infected with siRNA against Atm, PP5 or mock infection (−). (C) As in panel B; 48 hours after induction of Myc by OHT in the absence of ADR. (D) As in panel B; 24 hours after exposure to both ADR and OHT, or ADR or OHT alone plus the respective solvent, or left untreated.
- Figure S2. Growth regulation and immunoglobulin gene recombination in Atm−/− Eµ-myc lymphomas (JPG, 266 KB).
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(A) Spontaneous apoptosis analyzed by fluorescence-based TUNEL staining in cytospin preparations and in paraffin-embedded sections of control vs. Atm−/− lymphomas (n = 4); quantification (left) and representative examples (right). All quantitative data are mean ± SD; *P < .05; NS = not significant. (B) Proliferation measured as fraction of cells with S-phase DNA content (left); representative Ki67 immunostaining in lymphoma and nontransgenic spleen sections (right). Original magnification, ×200. (C) Immunoglobulin gene recombination status tested by multiplex genomic PCR to identify DJ and V(D)J joints (see “Materials and Methods”); representative examples of accomplished V(D)J recombinations in five Atm−/− lymphomas shown (out of 7 lymphomas per genotype tested, all accomplished V(D)J recombination); NIH3T3 fibroblasts as a negative control. (D) Representative cytogenetic analysis of an Atm−/− lymphoma (sample #9) by spectral karyotyping (SKY), demonstrating an unbalanced translocation between chromosomes 5 and 12, and a deletion of band D-F1 of chromosome 3. (E) Summary of all lymphomas evaluated by SKY (seven individual lymphomas per genotype with a total of 10 to 15 metaphases per case analyzed). Nonrecurrent translocations were found in six Atm−/− compared to two control samples. Note translocations involving the immunoglobulin (Ig) heavy chain locus IgH on chromosome 12 in three Atm−/− lymphomas compared to one control sample, and the recurrent interstitial chromosomal 3D-F1 deletion associated with the Atm−/− genotype in Eµ-myc lymphomas (6/7 Atm−/− compared to 0/7 control samples tested).
- Figure S3. NAC attenuates a Myc-induced PP5/Atm-governed DDR without affecting ARF (JPG, 119 KB).
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(A) p53 and p53-P-S18 levels in the presence and absence of NAC (15 mM) in OHT-treated MycERTAM-3T3 cells stably infected with siRNA against Atm, PP5 or mock infection (−) with Tubulin as a loading control. (B) ARF expression in MycERTAM-MEFs stably infected with an siRNA against Atm or an empty vector control analyzed after a 3-day exposure to 1 µM OHT and 15mM NAC as indicated (DAPI as a nuclear stain; blue). Original magnification, ×400. (C) ARF expression by RT-PCR (cells as in panel B, TBP as an internal control). (D) ARF expression levels by RT-PCR in pre-selected (ie, with no apparent deletions at the INK4a/ARF locus tested by multiplex genomic PCR) Eµ-myc lymphomas of the indicated genotypes; ARF−/− lymphoma as a control.
- Figure S4. Drug-induced Atm-dependent apoptotic defect in preneoplastic Eµ-myc lymphocytes (JPG, 80.8 KB).
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Trypanblue-based cytotoxicity analysis of preneoplastic Atm+/+ and Atm−/− Eµ-myc B cells measured at 19 hours of ADR (0.3 µg/mL) exposure in vitro (n = 3 individual samples per group). All quantitative data are mean ± SD.
- Figure S5. NAC cotherapy does not attenuate ADR-induced cell death (JPG, 78.3 KB).
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Eµ-myc lymphomas of the indicated genotypes (n = 4 each) were simultaneously exposed to increasing amounts of ADR (0 vs. 0.05 vs. 0.2 µg/mL) and to NAC (0 vs. 5 vs. 10 mM), and viability was measured after 19 hours by 7-amino-actinomycin D-flow–based analysis with untreated cells normalized to 100%. All quantitative data are mean ± SD.
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