F9CH γ-carboxylation in HEK-C3 cells is vitamin K dependent and inhibited by VKOR antagonists. (A) Doxycycline-induced HEK-C3 cells were supplemented with vitamin K1 and assayed by flow cytometry for γ-carboxylated F9CH and DsRed as described. F9CH Gla and DsRed signals were calculated as percent of maximal, and corrected for background in naïve HEK-Tet-ON parental cell line immunostained for γ-carboxylated F9CH. Percent maximal Gla/DsRed (▪) is plotted. (B) Doxycycline-induced, vitamin K1–supplemented HEK-C3 cells cultured in increasing concentrations of R,S-warfarin were assayed by flow cytometry for γ-carboxylated F9CH (and DsRed fluorescence). F9CH Gla and DsRed signals were calculated as percent of maximal, and corrected for background in naive HEK-Tet-ON parental cell line immunostained for γ-carboxylated F9CH.