Extended Data Fig. 8: EGFR/Ras pathway is required to selectively increase wild-type cell proliferation after injury.
From: Injury prevents Ras mutant cell expansion in mosaic skin
a) Schematic representation of the EGFR signaling pathway. HrasG12V is constitutively active and therefore less dependent on upstream activation of EGFR. b) Western blot analysis of p-EGFR(phospho-Tyr1068) normalized on total-EGFR (n = 6 mice) and of p-ERK1/2(phospho-Thr202/Tyr204) and p-AKT(phospho-Ser473) normalized on total-ERK1/2 and total-AKT (n = 3 mice). Unpaired, two-tailed t-test. c) Mitotic figure quantification in tdTomato+ and tdTomato− areas in HrasG12V/+-mosaic without or with EGFR-Dominant Negative (DN) expression (EGFR-WT or EGFR-DN). n = 3 mice. Unpaired or Paired two-tailed t-test for comparison between different or the same groups of mice. d) Revisit images of the same area of the basal stem cell layer. (left) The increase of tdTomato+ area. n = 3 mice. Unpaired, two-tailed t-test. e) Initial percent tdTomato+ area in the first revisit of injured-HrasG12V/+-mosaic treated with vehicle (n = 3 mice) or Gefitinib (n = 4 mice). f) Epidermal preparation immunofluorescence for phospho-Histone3 in wild-type (left) and constitutive-p21null (right) at postnatal-day-25. g) Phospho-Histone3+ cell quantification in wild-type, constitutive-p21null with or without LSL-tdTomato at postnatal-day-25 (n = 3 mice). Unpaired, two-tailed t-test. h) Whole mount immunofluorescence for phospho-Histone3 in wild-type (left) and constitutive-p21null (right) at postnatal-day-25. Phospho-Histone3+ dermal cell quantification (n = 4 mice). Unpaired, two-tailed t-test. i) Initial percent tdTomato+ area in the first revisit of uninjured wild-type-mosaic (n = 3 mice), HrasG12V/+-mosaic (n = 3 mice) and constitutive-p21null-HrasG12V/+-mosaic (n = 4 mice). j) Keratin10+ cell quantification in tdTomato+ and tdTomato− areas in HrasG12V/+-mosaic and constitutive-p21null-HrasG12V/+-mosaic in uninjured(U) ears (n = 3 mice). Unpaired or Paired two-tailed t-test for comparison between different or the same groups of mice. Exact p-values reported on the figure. ns indicates not statistically significant. At least three independent areas of approximately 300 μm2 were analysed for each mouse (Methods). Data are represented as means and standard deviations. Scale bar, 20 μm.
