Secretion levels of FVIII and FV in different cell lines. (A) LMAN1 and MCFD2 levels in WT, HepG2^LMAN1−, HepG2^MCFD2−, 293T^LMAN1−, and 293T^MCFD2− cells, as well as HCT116^+/− and HCT116^−/− cells. (B) Conditioned media were collected from HepG2 cell lines 48 hours after a fresh medium change. A FV expression construct was transfected into the indicated 293T and HCT116 cell lines and conditioned media were collected 48 hours after transfection. FV levels were measured by ELISA. (C) A FVIII expression construct was transfected into the indicated cell lines and conditioned media were collected 48 hours after transfection. FVIII activity and antigen levels in conditioned media were measured. (D) An AAT expression construct was transfected into HCT116^+/− and HCT116^−/− cells, and conditioned media were collected 48 hours after transfection. AAT concentrations in conditioned media were measured by ELISA. All data presented are means of 3 independent experiments, and the error bars represent standard deviations. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.