Supplementary Materials

Supplementary Material for:

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma

Carl Koschmann, Anda-Alexandra Calinescu, Felipe J. Nunez, Alan Mackay, Janet Fazal-Salom, Daniel Thomas, Flor Mendez, Neha Kamran, Marta Dzaman, Lakshman Mulpuri, Johnathon Krasinkiewicz, Robert Doherty, Rosemary Lemons, Jacqueline A. Brosnan-Cashman, Youping Li, Soyeon Roh, Lili Zhao, Henry Appelman, David Ferguson, Vera Gorbunova, Alan Meeker, Chris Jones, Pedro R. Lowenstein, Maria G. Castro*

*Corresponding author. E-mail: mariacas{at}

Published 2 March 2016, Sci. Transl. Med. 8, 328ra28 (2016)
DOI: 10.1126/scitranslmed.aac8228

This PDF file includes:

  • Materials and Methods
  • Fig. S1. SB-responsive shATRX plasmids are cloned to explore the impact of ATRX on GBM development.
  • Fig. S2. Cells cotransfected with shp53, shATRX, and NRAS plasmids are characterized at 15 days after injection.
  • Fig. S3. SB-mediated transfected cells are distinct from ependymal cells in mice.
  • Fig. S4. Characterization of p53/NRAS/shATRX mice 7 days after injection shows tumor cells expressing GFAP and Nestin.
  • Fig. S5. p53/NRAS/shATRX mice at 21 days after injection show tumor cells expressing OLIG2 and Nestin.
  • Fig. S6. Moribund p53/NRAS/shATRX mice show tumor cells expressing OLIG2, Nestin, and pERK.
  • Fig. S7. p53/NRAS/shATRX mice show loss of GFAP expression by 15 days after injection.
  • Fig. S8. ATRX mutations do not cluster in SNF2/helicase domain in human glioma.
  • Fig. S9. Primary GBM cell cultures are generated from SB-induced mouse tumors.
  • Fig. S10. Telomere length measured by qPCR is not different in tumors with or without ATRX.
  • Fig. S11. ALT was assessed by c-circle assay with DNA from mouse tumors and neurospheres.
  • Fig. S12. Cells transfected with shATRX plasmid show reduction in NHEJ by flow cytometry.
  • Fig. S13. NHEJ pathway is characterized by IHC.
  • Fig. S14. HR pathway is characterized by IHC.
  • Fig. S15. Mismatch repair pathway is characterized by IHC.
  • Fig. S16. SB mouse tumor growth (without radiation treatment) is characterized by luminescence.
  • Fig. S17. Tumors with ATRX loss show reduction in pDNA-PKcs before and after radiation treatment.
  • Fig. S18. Mouse tumors with ATRX loss show increased expression of γH2A.X in vitro and in vivo.
  • Fig. S19. Detailed schematic shows proposed impact of ATRX loss on GBM progression.
  • Table S1. Animal models of glioma all have RTK-RAS-PI3K alterations.
  • Table S2. Mice injected with p53/NRAS/shATRX have faster-growing tumors.
  • Table S3. MSI rate is increased in p53/NRAS/shATRX tumors.
  • Table S4. IDH1 and TP53 mutations do not alter SNV rate calculated by two-way ANOVA model.
  • Table S5. Telomere qPCR average telomere length ratio worksheet and standard curve show similar results in all experimental groups.
  • Table S6. Immunofluorescence analysis shows reduced pDNA-PKcs expression in ATRX-deficient mouse GBM.
  • Table S7. Antibodies used for tissue analysis are summarized in table form.

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