A rabbit polyclonal antiserum specific for the nsp2 subunit of the SFV replicase was used as the primary antibody (6). Analysis selleck Nutlin-3a of SFV-LacZ infectivity and ��-galactosidase expression in vitro. BHK-21 or HCC-derived cell lines were infected in duplicate plates with serial dilutions of the same SFV-LacZ virus stock. Twenty-four hours later, cells from one dish were lysed and the amount of ��-galactosidase protein present in the lysate was measured as described previously (23). Cells from the duplicate dish were stained with 5-bromo-4-chloro-3-indolyl-��-d-galactopyranoside (X-Gal) (Invitrogen), and the number of cells infected with SFV-LacZ was determined. The amount of ��-galactosidase protein produced per cell was calculated for each virus dilution by dividing the amount of ��-galactosidase protein detected per dish by the number of SFV-LacZ-infected cells in each dish.
Analysis of luciferase and IL-12 expression in vivo. Four woodchucks with HCC were used to determine that the expression of luciferase and IL-12 in hepatic tumors is dependent on the SFV dose and to verify that intratumoral IL-12 expression mediates increases in the levels of type I and II IFNs. Animals were anesthetized by intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). A blood sample was obtained, followed by laparotomy to expose the liver. Vector SFV-Luc or SFV-enhIL-12, each in a total volume of 0.6 ml, was administered intratumorally into one hepatic tumor at 10 separate sites. The first woodchuck received 1 �� 109 vp of SFV-Luc, whereas the second woodchuck was administered a threefold-higher dose of SFV-Luc (i.
e., 3 �� 109 vp). The latter woodchuck also received 3 �� 109 vp of SFV-enhIL-12 into another hepatic tumor. The third woodchuck was administered a fourfold-higher dose of SFV-enhIL-12 (i.e., 1.2 �� 1010 vp). The fourth woodchuck was left untreated, and the hepatic tumor from this animal served as a control. Twenty-four hours later, woodchucks were euthanized, a blood sample was obtained, and tissues (hepatic tumors, adjacent liver tissues, and other organs) were excised and snap-frozen in liquid nitrogen. Following homogenization in lysis buffer (phosphate-buffered saline containing 0.05% [vol/vol] Tween20 and protease inhibitor cocktail tablets [Complete; Roche, Basel, Switzerland]), samples were centrifuged twice for 10 min at 9,300 �� Entinostat g. Luciferase activity in the supernatants was determined using a conventional luminometer. IL-12 concentrations were determined in supernatants and in serum using a mouse p70-specific enzyme-linked immunosorbent assay kit (BD Bioscience, Franklin Lakes, NJ). The amount of total protein present in tissue homogenates was quantified using a standard Bradford assay.