Reconstitution of PAR2 signaling in PAR2^−/− PyMT breast cancer cells. (A) Growth properties of PyMT WT and PAR2^−/− breast cancer cells after in vivo passage through the mammary fat pad (n = 8 mice/group, representative of at least 2 experiments). (B-C) PyMT PAR2^−/− cells were transduced twice with control (Mock), PAR2^WT, or PAR2^ΔARR retrovirus. (B) Quantification of transduction efficiency was determined by flow cytometry measuring DS-Red fluorescence. PAR2-reconstitution did not change TF cell surface expression. (C) PAR2 reconstitution restores CXCL1 induction by PAR2 agonist SLIGRL (100μM), trypsin (10nM), VIIa (5nM), or the ternary complex (VIIa (5nM), X (50nM), nematode anticoagulant protein c2 (200nM)). Hirudin (200nM) was added to all reactions that included coagulation factors. CXCL1 induction was assessed by quantitative reverse transcription polymerase chain reaction after 90 minutes of stimulation. Means and ± SD, n = 4; * P < .05 different from mock-transduced cells by ANOVA.