Figure 4. Loss of Nrf2 phenocopies Atg7 deletion and is associated with increased lifespan.

A. Representative IHC staining for p62 and NRF2 in lung tumors of Braf^V600E/+; Atg7^+/+ and Braf^V600E/+; Atg7 ^−/− mice at the indicated time post Cre. Scale bar=10 μm. Quantification is shown below. Error bars are SEM.

B. Western blot analysis of NRF2 accumulation in lysates of tumors harvested from Braf^V600E/+; Atg7 ^+/+ and Braf^V600E/+; Atg7^−/− mice 10 weeks post Cre. Numbers refer to individual animals. A blank lane was intentionally included between the normal lung and tumor lysates. An additional Atg7 null tumor lysate was spliced out for aesthetic reasons.

C. H&E staining of single lung lobes showing dramatic increase in size and number of tumors in Nrf2^−/− mice independent of autophagy status 4 weeks post Cre. Scale bar= 3 mm. Quantification of tumor burden shown at right. The Nrf2^+/+ and Nrf2^−/− graph is the aggregate of autophagy competent and deficient braf tumors. These data are plotted individually in the next graph. The difference in tumor burden between the Nrf2 ^−/−;Braf ^V600E/+; Atg7^+/+ and Nrf2 ^−/−;Braf ^V600E/+; Atg7^−/− mice is not statistically significant (p=0.63). The difference in tumor burden between the Nrf2 ^+/+;Braf ^V600E/+; Atg7^+/+ and Nrf2 ^+/+;Braf ^V600E/+; Atg7^−/− mice is not statistically significant in this experiment (p=0.49). Please refer to similar experiments in Figs. 2A–C, S3, and 3B.

D. Representative images of H&E and IHC analysis of γ-H2AX 4 weeks post Cre. Scale bar=10μm

E. Representative images of IHC for 8-oxo-dG 17 weeks post Cre. Scale bar= 10μm

F. Kaplan Meier analysis of overall survival of the Nrf2; Braf; Atg7 compound mutant mice. The number of mice per group and median survival are indicated. Note that the increased survival between the Nrf2^−/−;Braf ^V600E/+; Atg7^+/+ and Nrf2 ^+/+;Braf ^V600E/+; Atg7^+/+ mice just misses statistical significance (p= 0.06).