(A) Lentiviral-based vector allowing for constitutive expression of a myc-tagged ETS2 cDNA, either WT or harboring silent mutations that abrogate shRNA binding. Immunoblot analysis to verify that both constructs express ETS2 protein at equivalent levels in HRasG12V; Tgfbr2-null keratinocytes, but that when Ets2#649 shRNA is transduced, myc-ETS2 is only suppressed in cells transduced with the WT 3’UTR and not the mutant 3’UTR version. (B) Vector allowing for Doxycycline-inducible expression of a myc-tagged ETS2 cDNA, either WT or harboring the phosphomimetic T72D varient. H2B-RFP, driven by a constitutively active PGK promoter, was inserted to control for lentiviral transduction. When the vector is transduced in cells or mice expressing the rtTA Doxy-inducible transactivator, Doxy will activate the Myc-tagged, ETS2-T72D protein. Immunoblot analysis to verify ETS2 protein expression in HRasG12V; Tgfbr2-null keratinocytes. (C) Ectopic induction of wild-type ETS2 in skin epidermis does not disrupt the morphology or function of the tissue. At E9.5, litters harboring K14rtTA and control embryos were infected in utero with lentivirus harboring the wild-type ETS2 construct in (B). This method allows for efficient and selective transduction of the skin epithelium by E12.5. Postnatally, these pups were administered Doxycycline to activate rtTA (if present) and 4 weeks later, their skin was analyzed for transduction (H2BRFP) and epidermal architecture (K5 and K10). Sagittal sections of skin are stained with DAPI. Note no significant differences between the control and the WT ETS2 expression. (D) Same experiment in (C) but with the ETS2 (T72D) construct in (B). Note striking difference in phenotype when this version of ETS2, constitutively activated at its Ras/MAPK phosphorylation site, is expressed in otherwise normal skin epidermis. Immunolabeling is for β4 integrin to mark the basal surface of cells attached to an underlying basement membrane; Ki67, to mark actively cycling cells; and CD31, an endothelial marker for blood vessels. Quantifications of Ki67 and blood vessels are shown beneath the immunofluorescence pictures. Bars = 100 µm. (E) FACS profiling of the cells for CD11b, a pan marker of inflammatory immune cells. FACS, fluorescence-activated cell sorting.