0, then heated in a microwave, placed in a steamer for 30 minutes, and steadily cooled third down on the bench top for 20 minutes. Sections were incubated with a blocking buffer including 5% donkey serum plus 1% bovine serum albumin in PBS�C0.3% Triton X-100 for 1 hour, followed by treatment with mouse Ig blocking reagent (Vector Laboratories) for 1 hour at room temperature. Sections then were incubated with a mouse monoclonal anti�C��-SMA (1:1000; Sigma-Aldrich) at room temperature for 1 hour. After washing three times with TBST for 5 minutes each, sections were incubated with Alexa Fluor 555 donkey anti-mouse IgG (1:500; Invitrogen, Grand Island, NY) for 30 minutes at room temperature. After these processes, TUNEL staining was performed using a commercial kit (In Situ Cell Death Detection Kit; Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature.

After washing three times with TBST for 5 minutes each, sections were mounted with DAPI-containing media (Invitrogen). Images were taken with a fluorescent microscope (Eclipse E800; Nikon) and recorded using Openlab3 software version 5.5.2 (PerkinElmer, Waltham, MA). Isolation of HSCs Primary HSCs were isolated from WT and Nogo-B KO mice by in situ perfusion of livers with pronase-collagenase, followed by density gradient centrifugation using Nycodenz (Histodenz; Sigma-Aldrich) density gradients as described.36,37 HSCs were cultured in Dulbecco��s modified Eagle��s medium with high glucose (DMEM; Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 ��g/mL streptomycin, and 1% l-glutamine in humidified air containing 5% CO2 at 37��C.

HSCs were cultured for more than 14 days to fully transform to MF-HSCs.38 Treatment of MF-HSCs with STS, FAS Ligand, and Tunicamycin MF-HSCs were seeded in 6-well tissue culture plates at a density of 2.0 �� 105 cells per well and incubated in humidified air containing 5% CO2 at 37��C overnight. Media was changed to serum-free conditions in DMEM for 24 hours, and MF-HSCs were treated with 1 ��mol/L staurosporine (STS; EMD Millipore, Billerica, MA) for 0, 2, 4, 6, 8, and 10 hours to induce apoptosis. To determine whether apoptosis was Fas-receptor�Cdependent, MF-HSCs were treated with 50 ng/mL Fas ligand (Calbiochem) in the presence of 20 ng/mL cycloheximide (Calbiochem) for 10 hours.

To induce ER stress, MF-HSCs were treated with tunicamycin at concentrations of 0, 0.5, 1, 2, 5, and 10 ��mol/L for 24 hours. Hepatocyte Isolation Hepatocytes were isolated from WT and Nogo-B KO mice by collagenase perfusion as previously described39 with slight modifications. Briefly, Drug_discovery cells were cultured on collagen-coated cell culture dishes or glass coverslips in Hepatocyte Maintenance Medium (Clonetics/Lonza, Basel, Switzerland) supplemented with Hepatocyte Maintenance Medium SingleQuots (Clonetics/Lonza) and Matrigel (BD Biosciences, San Jose, CA).