The media was subsequently collected after 1 or 2 hours. AML12 mouse hepatocytes (ATCC) were grown in DMEM/F12 media supplemented with 10% FBS and ITS (Invitrogen). Cells were pretreated with 200 ng/mL of actinomycin D (ActD) for 30 minutes. Media was then changed to 150 μL of conditioned Kupffer cell media plus 200 ng/mL ActD. After 18 hours, media was removed and cells were fixed. Cells were quantitated by crystal violet assays and hepatocyte number was calculated based on a standard curve.19
TNF-α antibody (AB-410-NA) was from R&D Systems. Primary hepatocytes were pretreated with 200 ng/mL of ActD in Williams E media + 5% FBS for 30 minutes. Media was then changed to include increasing amounts of recombinant mouse TNF-α (Peprotech, Rocky Hill, NJ) plus ActD. After 18 hours, media was removed and cells were fixed and quantitated by crystal violet assays. Metformin order Cells were collected and homogenized in 2× Laemmli Buffer. For HGFL determination, cells were cultured in serum-free media and the media was collected after 36 hours. Media was concentrated with Amicon Ultra-4
centrifugal selleck chemicals llc filters (Millipore, Billerica, MA). Protein concentrations were determined using the Micro BCA Kit (Pierce Biotechnology, Rockford, IL). Primary antibodies used were anti-NF-κB p65, anti-pNF-κB p65 Ser536, anti-pIKKα/β, anti-IKKβ anti-Caspase-3 (Cell Signaling, Boston, MA), anti-HGFL (T-19, Santa Cruz Biotechnology, Santa Cruz, CA). Mice were injected with 0.8 μg LPS and 30 mg GalN in saline and normalized to 30 g body weight Resminostat (500 μL total volume) or GalN alone. This low dose of LPS does not alter mortality in the Ron TK-deficient mice, but when combined with GalN induces significant liver injury.7, 16 Blood was collected and plasma alanine aminotransferase
(ALT) levels were determined at Shriners Hospital. Paraffin-embedded sections of liver tissue were analyzed by TUNEL staining.16 For each liver tissue section per mouse, the number of TUNEL-positive cells in three random high-powered fields was counted by an investigator blinded to treatment group. Statistical significance for all analyses was determined by Student’s t test, Logrank, or one-way analysis of variance (ANOVA) using GraphPad Prism 3.03 software (La Jolla, CA). Error bars represent standard error of the mean (SEM). Quantitative data directly comparing Ron expression in liver macrophages (Kupffer cells) and in liver parenchymal cells in the mouse are lacking. To examine Ron expression in mouse Kupffer cells and hepatocytes, populations of murine Kupffer cells and hepatocytes were collected. The isolated Kupffer cells ranged in purity from 90%-95% following centrifugal elutriation based on F4/80 immunostaining (Fig. 1A) and the ability of the cells to ingest fluorescent beads (data not shown). Hepatocyte identity was confirmed with albumin immunostaining and purity was over 99% (Fig. 1A).