Figure 8. p62 mediates lycopene-induced keap1 degradation and cutaneous tumor prevention. (A) Time-dependent (bottom) and dose-dependent (top) study of p62 protein levels. Cells were pretreated with 8 μM lycopene for different times or with different doses of lycopene for 6 hours and then exposed with TPA for additional 2 hours, and the p62 was analyzed by western blot. (B) Cells were treated with lycopene for 4 hours and then exposed with TPA for additional 1 hours. Co-IP was done as described in Methods, and the immunoprecipitants were immunoblotted using antibodies for keap1, p62 and GAPDH. (C) Cells were transfected with shRNA specific for p62 or a nonspecific control shRNA for hours and then treated with lycopene (8 μM) for 6 hours and then TPA for 2 additional hours. Keap1 and p62 proteins were assayed by Western blot. (D) Inhibitory effect of lycopene pretreatment on the TPA-induced transformation of shMock- and shp62-transfected JB6 P+ cells. (right) Quantitative analysis of this soft agar assay (n=3). The data are presented as the mean ± SD. **p < 0.01, ***p < 0.001 (versus TPA alone). The results are representative blot image of three independent experiments shown in A, B, C, respectively.