Supplemental Data

Colorado Denver. The inducible head-and-neck specific knockout system consists of two mouse lines, the K14.CrePR1 or K5.Cre*PR1 mice, in which Cre recombinase can be activated in head-and-neck epithelia by RU486 (1, 2), and the Smad4f/f mice, in which the Smad4 gene is floxed (3). These mouse lines were crossbred to generate compound mice, allowing for homozygous Smad4 deletion (Figure S1A). Littermates were genotyped at 3 weeks of age using primer pairs P9 and P10, and grouped based on genotypes for the experiments. RU486 (100 l of 0.2g/l in sesame oil) was applied in the oral cavity of 4-week-old bigenic mice daily for 5 consecutive days to induce homozygous deletion of the Smad4 gene. Monogenic (K14.CrePR1, K5.Cre*PR1, or Smad4f/f) control littermates were treated with the same RU486 regimen as bigenic mice. To generate control tumors, we utilized an RU486-inducible K15.CrePR1 line (4) in combination with a knock-in mutant Kras G12D allele (5) to generate K15.CrePR1/Kras G12D mice. We also mated these mice to Smad4f/f mice to generate