Data Supplement from Targeted Deletion and Lipidomic Analysis Identify Epithelial Cell COX-2 As a Major Driver of Chemically Induced Skin Cancer

Skin F4/80+ macrophage from Cox-2deltaM (Cox-2flox/flox;LysMCre+) mice undergo effective targeted Cox-2 gene deletion. Macrophages were isolated from the skin of 7-9 week old Cox-2deltaM and Cox-2fl/fl mice that were treated one time with TPA to induce COX-2 expression. Skin specimens were treated with 3 mg/ml Dispase for 16 hrs at 4oC; the epidermis was then separated from the dermis. The dermal layer was further digested, at 37 oC for 1.5 hrs, in RPMI1640 medium containing 1mg/ml collagenase I (Invitrogen, Carlsbad, CA). The dissociated cells were stained with PE-conjugated anti-F4/80 antibody, and F4/80+ cells were isolated by FACS. (A) FACS plots show, in red, the sorted F4/80+ populations from Cox-2deltaM and Cox-2fl/fl dissociated dermal layers. (B) DNAs from the isolated F4/80+ cell populations were extracted and the presence of the Cox-2fl, Cox-2del, and Cre alleles were examined by PCR. PCR analysis demonstrates the presence of both the Cre and the Cox-2del alleles in F4/80+ cells isolated from Cox-2deltaMmice. (C) Total RNA was isolated with an RNeasy Mini Kit (Qiagen, Valencia, CA), from sorted F4/80+ cell populations. RNAs were reverse transcribed into cDNA with SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen, Carlsbad, CA), and quantitative PCR was done in the iQ thermal cycler (Bio-Rad) using the iQSYBR Green Supermix (Bio-Rad). Cox-2 forward primer: 5'-TGA TCGAAGACTACGTGCAA-3'; reverse primer: 5'-GTGAGTCCATGTTCCAGGAG-3'. The graph shows significantly decreased Cox-2 gene expression in F4/80+ Cox-2deltaM cells when compared to F4/80+ Cox-2fl/fl cells. β-Actin was used as the reference gene. Error bars are SD. * indicates p<0.05.