Ets1 in Squamous Neoplasia Figure 9. Ets1 expression induces upregulation of MMP13. (A) Immuno-histochemical staining for MMP13 (brown) in the skin of wild-type and BT mice. Sections were counterstained with hematoxylin. Note expression of MMP13 in the invading front (hair follicles and sebaceous glands) and in hyperplastic keratinocytes in the BT sample. (B) Western blot for MMP13 expression in skin extracts of wild-type and BT mice. GAPDH is a loading control. (C) Luciferase assays using different lengths of the mouse MMP13 promoter (MMP13) or an empty vector lacking a promoter (pGL3B) transfected into mouse MK keratinocytes. The MMP13 promoter was transfected with an expression vector driving HA-tagged mouse Ets1 (pCMV-HA- Ets1) or with a control empty vector (pCMV-HA). Results represent the fold induction of luciferase activity in cells transfected with the Ets1 expression vector as compared to empty vector and have been corrected for the activity of the internal control plasmid (pCMVb-gal). Arrows in the p391 construct indicate the positions of primers used in part D for chromatin immunoprecipitation. (D) Chromatin immunoprecipitation assays to detect association of Ets1 with the endogenous MMP13 promoter in wild-type and BT mice. Antibodies used for immunoprecipitation include anti-Ets1 or a control rabbit IgG and anti-HA or a control rat IgG. Immunoprecipitated chromatin was amplified with primers specific for the mouse MMP13 promoter or for the GAPDH gene. Results represent relative concentrations of products amplified by qPCR. doi:10.1371/journal.pone.0004179.g009 PLoS ONE | www.plosone.org 10 January 2009 | Volume 4 | Issue 1 | e4179