Fig 2: Fra-2 is phosphorylated and stabilized by ERK1/2 upon mKC differentiation. (A) p-ERK1/2 immunohistochemistry in the skin of wild-type embryos at E17.5. Arrows point toward suprabasal expression, and the dashed white line indicates basement membrane. n = 3. (B) ERK1/2 activation shown by p-ERK1/2 Western blot during in vitro mKC differentiation (0–72 h Ca2+). n = 3. (C) Immunoprecipitation of Fra-2 during mKC differentiation (0–48 h Ca2+) and Western blot analyses of Fra-2 and p-Fra-1/2. n = 3. (D) Western blot analysis of Fra-2 upon Ca2+ treatment (48 h) and ERK1/2 inhibition with FR180204. n = 3. (E) Immunoprecipitation of Fra-2 upon Ca2+ treatment (48 h) and ERK1/2 inhibition with FR180204 and Western blot of Fra-2 and p-Fra-1/2. n = 3. (F) mRNA expression analyses of EDC genes upon Ca2+ treatment (48 h) and ERK1/2 inhibition with FR180204. n = 3; (*) P < 0.05; bars represent mean ± SD. (G) Fra-2 protein expression of primary mKCs infected with lentiviruses expressing Fra-2 wild type (Fra-2 WT) or mutant Fra-2 deficient for C-terminal phospho-acceptor sites (Fra-2 S320A/T322A) compared with empty vector-infected cells. n = 3. (H) Differential Fra-2 and EDC gene expression of primary mKCs expressing Fra-2 wild type or Fra-2 S320A/T322A compared with empty vector-infected cells. n = 3. (I) Schematic model of EDC gene regulation by Fra-2, Ezh2, and ERK1/2: In basal cells, methylated Fra-2 (Fra-2-Me) bound at TPA-responsive elements (TREs) of EDC gene promoters, and interacting with Ezh2 prevents EDC gene expression. Upon mKC differentiation, Fra-2 becomes phosphorylated (Fra-2-P) by ERK1/2 and loses its methylation, and Ezh2 protein levels and H3K27 methylation at TSSs of EDC promoters are reduced, which together induce EDC gene expression.

Fig 3: Fra-2 induces terminal epidermal differentiation. (A) Fra-2 mRNA expression during Ca2+-induced in vitro mKC differentiation. K1 and Flg indicate induction of early and terminal epidermal differentiation markers. n = 3; (*) P < 0.05; bars represent mean ± standard deviation (SD). (B) Fra-2 protein expression during Ca2+-induced in vitro mKC differentiation. Loss of ?Np63 indicates induction of differentiation. n = 4. (C) Fra-2 mRNA expression of basal (a6 integrin high) and differentiated (a6 integrin low) mKCs isolated from newborn pups. K1 and Flg indicate induction of early and terminal epidermal differentiation markers. n = 3; (*) P < 0.05; bars represent mean ± SD. (D) Fra-2 IF of wild-type skin at E17.5. n = 6; 40× magnification. (E) Differential EDC gene expression of Flg, Lor, Tchhl1, Lce1k, and Fra-2 expression in epidermal samples of mutants ectopically expressing Fra-2 (Fra-2Ep-tetOFF) compared with control littermates at E17.5. n = 6 per genotype; (*)P < 0.05; bars represent mean ± SD. (F) Basal cell marker expression (K5, green) and late differentiation marker expression (Lor, red, top panel; Flg, red, bottom panel) in Co and Fra-2Ep-tetOFF skin at E17.5. n = 6 per genotype; 40× magnification. White bars indicate the thickness of differentiated epidermal layers, which is quantified in Supplemental Figure S1C. (G) Differential EDC gene expression and Fra-2 expression in primary Fra-2Ep-tetOFF mKCs compared with control mKCs under basal conditions and upon Ca2+ treatment for 24 h in vitro. n = 3; (*) P < 0.05; bars represent mean ± SD. (H) Macroscopic appearance of SOS+ and Fra-2Ep-tetOFF SOS+ papillomas at 5 wk of age. n = 6 per genotype. (I) Analyses of SOS+ and Fra-2Ep-tetOFF SOS+ papillomas. H&E (top panel), basal cell marker expression (K5, green) and late differentiation marker expression (Lor, red, middle panel; Flg, red, bottom panel) in SOS+ and Fra2Ep-tetOFF SOS+ papillomas at 5 wk of age. 20× magnification. The insert depicts 40× magnification. Lor and Flg mean fluorescence intensity is quantified in Supplemental Figure S1D. n = 6 per genotype.

Fig 4: Fractionated radiation up-regulates Notch signaling through activation of STAT3(A) Western blot analysis for STAT3, NF-κB and Fra-1 phosphorylation status rafter irradiation of MCF7 breast cancer cells. (B) qRT-PCR for Notch2, Jagged-1 and DLL4 mRNA levels after irradiation of MCF7 cells that are transfected with siRNA targeting STAT3, NF-κB and Fra-1. (C) qRT-PCR for Notch2, Jagged1 and DLL4 mRNA levels after irradiation of MCF7 cells that are treated with STAT3 inhibitor (2 μM). (D) Western blot for NICD2, Jagged1 and DLL4 after fractionated irradiation (2 Gy × 3) of MCF7 that are treated with STAT3 inhibitor (2 μM). Error bars represent mean ± S.D. of triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001.