Extended Data Fig. 10. Injury-repair or p21loss specifically increases phospho-ERK1/2 in wild-type cells.
a) Western blot analysis of p-ERK1/2(phospho-Thr202/Tyr204) normalized on total-ERK1/2. b) Western blot analysis of p-AKT(phospho-Ser473) normalized on total-AKT. (a, b) wild-type (n = 4 mice), HrasG12V/+-mosaic (n = 3 mice), HrasG12V/+-max (n=3 mice) and constitutive-p21null (n = 3 mice). Unpaired, two-tailed t-test. Data are represented as means and standard deviations. c) Confocal representative images of the epidermal preparation immunofluorescence for p-ERK1/2 and phalloidin in HrasG12V/+-mosaic. (left) Insets of p-ERK1/2+ cells: a) cells in interphase b) mitotic cell and c) footprint of a cell that is departing from the basal layer. d) Quantification of p-ERK1/2+ cells in tdTomato+ and tdTomato− areas in HrasG12V/+-mosaic in injured/3 days PWI(I) and uninjured(U) ears. At least four independent 150 μm2 areas were analysed for each mouse. (c, d) n = 3 mice. Unpaired or Paired two-tailed t-test for comparison between different or the same groups of mice. ns indicates not statistically significant. Data are represented as means and standard deviations. Scale bar, 20 μm. (a, b, c, d) Exact p-values reported on the figure. e) Final model. In the uninjured mosaic skin epidermis, Ras cells integrate and expand, outcompeting wild-type neighbors. During injury-repair of mosaic skin, the competitive advantage of Ras cells is suppressed, and oncogenic growths do not develop. EGFR signaling pathway is crucial for this selective increase of wild-type cell divisions that prevents Ras cell expansion during injury-repair. Inducing proliferation, via constitutive p21 loss, mimics the injury condition in uninjured skin, counteracting the competitive advantage of Ras cells. Our data support a model whereby injury-repair and p21 loss increase the activity of ERK1/2, a Ras downstream pathway that controls cell proliferation. This leads to comparable ERK1/2 levels between wild-type and Ras cells, resulting in an increase of dividing wild-type cells that effectively prevent Ras mutant cell expansion.