12 o tetradecanoyl phorbol 13 acetate tpa

12-O-tetradecanoylphorbol-13-acetate (TPA) and okadaic acid (OA) were purchased from LC Laboratories (Woburn, MA). Etomoxir ethyl ester (Eto) was purchased from US Biological (Salem, MA). Trypsin, Trypsin-EDTA, Keratinocyte Serum Free Media (KSFM), Hank’s Balanced Salt Solution (HBSS), dispase, gentamicin, fungizone, tetramethylrhodamine methyl ester (TMRM), MitoTracker Green (MTG), and Lipofectamine 2000 were purchased from Life Technologies (Grand Island, NY). Acetone, 7,12-dimethylbenz[a]anthracene (DMBA), and all other reagents (unless otherwise noted) were purchased from Sigma (St. Louis, MO).

BK5.Cre mice (30 (link)) were bred to Twist1^flox/flox mice (31 (link)) to generate offspring carrying floxed Twist1 alleles [BK5.Cre x Twist1^flox/flox (Twist1 KO)]. BK5.Cre x Twist^+/+ (WT) were used as controls for the experiments as indicated. Adult transgenic and non-transgenic littermates were used for indicated experiments at 6–8 weeks of age unless otherwise noted. The dorsal skin of each mouse was shaved 48 hours prior to the first chemical treatment. 7,12-dimethylbenze[a]anthracene (DMBA) (Sigma-Aldrich, Milwaukee, WI, USA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (LC Laboratories, Woburn, MA, USA) solutions were prepared in reagent grade acetone (Ace) and applied topically to the dorsal skin in a total volume of 200 μl. For two-stage skin carcinogenesis experiments, mice were initiated with a single topical application of 25 nmol DMBA. Two-weeks following initiation with DMBA, 13.6 nmol TPA was applied topically twice-weekly for the duration of the experiment. The incidence and numbers of both papillomas and SCCs were tabulated weekly.

7,12-dimethylbenz(a)anthracene (DMBA) (≥ 95% purity), N6, 2’-O-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) and Sodium orthovandate (Na3VO4) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was obtained from LC laboratories (Woburn, MA). Rat anti-mouse IL-12Rβ2 and IL-23R were purchased from R&D; Rabbit anti-mouse VEGF, TRP2, Mouse anti-human S100, Rat anti-mouse vimentin were obtained from Santa Cruz Biotechnology, Inc. Rat anti-mouse pERK was from BD biosciences. CD4-PE, CD4-FITC, FOXP3-PE, FOXP3-v450, IA/IE-FITC, IL-17-Percp-Cy5.5, IL-10-PE, CD45.2-Percp-Cy5.5, CD45.2-FITC were obtained from eBiosciences. IFNγ-PE-Cy7, CD8-Alexa-647, and CD8-PE were obtained from BD-pharmingen.