Methods in Supplementary Information / Takaoka et al.
Conditions of IFN-treatment, virus infection and X-ray irradiation

Each assay was carried out under the experimental conditions as described in the main article. Unless mentioned otherwise, the concentration of IFN- used in the assays is 500 U/ml. The M.O.I. of VSV used in the assays; 1.0. The dose of X-ray irradiation; 30 Gy.

EMSA

EMSA was performed as described in the main text. Competition assay was conducted with different concentrations of each non-radiolabeled oligonucleotide probe containing the element of h-p53-ISRE1 or h-p53-ISRE2, as well as those of h-p53-ISRE1-mutand h-p53-ISRE2-mut, which havemutated sequences in the putative ISREs: h-p53-ISRE1; -1399 GGGAGTGGAGAGAGAAACTGGGTCC -1374, h-p53-ISRE2; +829 ATTTCCGGTTTCTTTTTGCCGGAGC +854, h-p53-ISRE1-mut; GGGAGTGGAGGGAATGGCTGGGTCC, h-p53-ISRE2-mut; ATTTCCGGCCATTTCTTGCCGGAGC.

RT-PCR analysis

Total RNA from MEFs was reverse-transcribed and PCR-amplified as described previously1,2. The primers used for RT-PCR were as follows: human p53, 5’-GCCCAACAACACCAGCTCCT (sense) and 5’-CCTGGGCATCCTTGAGTTCC-3’(antisense); human GAPDH, 5’-GAGTCAACGGATTTGGTCGT-3’(sense) and 5’-GACAAGCTTCCCGTTCTCAG-3’(antisense); HPV16 E6, 5’-GCAACAGTTACTGCGACGTG-3’(sense) and 5’-GGACACAGTGGCTTTTGACA-3’(antisense); mTERT, 5’-CACATTCCAGAAGAACAGG-3’(sense) and 5’-CAGATGGGCATGGCTAG-3’(antisense). Quantitative real-time RT-PCR was performed as described in Fig. 2b of the main article.

RNA blot analysis

A hybridization probe for Bax mRNA was generated by PCR amplification of a full-length of the coding region of the mouse Bax cDNA. Probes for p21WAF1/Cip1 or OAS mRNA were described previously3,4.

Expression of E6, E1A, wild-type p53 or mutant p53 via retroviral gene transfer

pLRHL-16E6, a retroviral expression vector,was used for theexpression of HPV16 E6 protein5. Adenovirus 2 E1A 12S cDNA, which was derived from pHApr-E1A12s (Ref. 6), was inserted into the blunted EcoRI site of pBabe-puro, a retrovirus expression vector. Wild-type p53 cDNA or mutant p53 cDNA, respectively derived from pSVp53c17 or pLSVNc-9 (Ref. 7), was ligated to the BglII-SmaI cloning sites of pBabe-puro. With the resultant retroviral expression plasmids, pBabe-E1A, pBabe-wt-p53 or pBabe-mt-p53, the retrovirus-mediated gene transfers were performed as described in Fig. 3 of the main article.

Cytopathic effect (CPE) assay

CPE assay with VSV (1.0 M.O.I.) or EMCV (0.1 M.O.I.) was performed as previously described8. The results for each assay were confirmed by triplicated experiments which were separately conducted with at least two independent clones of MEFs derived from the same littermates of the wild-type or mutant mice.

References in Supplementary Information

1.Gudjonsson, T. et al. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties. Genes Dev.16, 693-706 (2002).

2.Drissi, R., Zindy, F., Roussel, M. F. & Cleveland, J. L. c-Myc-mediated regulation of telomerase activity is disabled in immortalized cells. J. Biol. Chem.276, 29994-30001 (2001).

  1. Tanaka, N. et al. Cooperation of the tumour suppressors IRF-1 and p53 in response to DNA damage. Nature382, 816-818 (1996).
  2. Sato, M. et al.Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-/ gene induction. Immunity13, 539-548 (2000).
  3. Kiyono, T. et al. Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein. Proc. Natl. Acad. Sci. U.S.A.94, 11612-11616 (1997).
  4. Nakajima, T. et al. Suppression of adenovirus E1A-induced apoptosis by mutated p53 is overcome by coexpression with Id proteins. Proc. Natl. Acad. Sci. U. S. A.95, 10590-10595 (1998).
  5. Eliyahu, D. et al. Meth A fibrosarcoma cells express two transforming mutant p53 species. Oncogene3, 313-321 (1988).
  6. Takaoka, A. et al. Cross talk between interferon- and- signaling components in caveolar membrane domains. Science288, 2357-2360 (2000).