Nogo-B Knockdown in Human Hepatic Stellate Cell Line (LX2) LX-2 cells were a kind gift from Dr. Scott L. Friedman (Mount Sinai School of Medicine, selleck chemical Erlotinib New York, NY).40 Cells were seeded on 6-well tissue culture plates at a density of 1.5 �� 105 cells per well and incubated in humidified air containing 5% CO2 overnight at 37��C. Cells were transfected with 100 nmol/L Nogo-B small-interfering RNA in 750 ��L Opti-MEM (Gibco, Invitrogen) with 2 ��L Oligofectamine (Invitrogen) and incubated for 6 hours. Then, 750 ��L of DMEM containing 20% FBS and 2% l-glutamine was added to each well. After 48 hours of incubation, these cells were starved in DMEM without FBS for 24 hours and treated with 100 nmol/L STS for 0, 2, 4, 6, 8, and 10 hours. Nogo-B Overexpression in LX2 LX2 cells were seeded in 12-well tissue culture plates at a density of 1.

0 �� 105 cells/well and incubated in humidified air containing 5% CO2 overnight at 37��C. Cells were transfected with 0.5 ��g of hemagglutinin-tagged Nogo-B plasmid (or pcDNA3 vector alone as a negative control) in 500 ��L Opti-MEM (Gibco, Invitrogen) with 1.5 ��L FuGENE 6 (Roche Diagnostics) and incubated for 6 hours. Then, 500 ��L of DMEM containing 10% FBS and 1% l-glutamine was added to each well. After 48 hours of incubation, cells were starved in DMEM without FBS for 24 hours, followed by treatment with 100 nmol/L STS for 4 and 8 hours, respectively. For immunocytochemistry, cells were washed with cold PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. After washing three times with PBS for 5 minutes each time, cells were incubated in a buffer containing 0.

3% Triton X-100, 5% donkey serum, and 1% bovine serum albumin in PBS at room temperature for 1 hour. After washing with PBS, cells were incubated with antibodies including rat anti-HA (1:1000; Roche Diagnostics) and rabbit anti-cleaved caspase-3 (1:1000; Cell Signaling), which were diluted with 1% bovine serum albumin in PBS overnight at 4��C. Cells then were incubated with Alexa Fluor 488 donkey anti-rat IgG (1:500; Invitrogen) or Alexa Fluor 588 donkey anti-rabbit IgG (1:500; Invitrogen) as a secondary antibody for 1 hour at room temperature. Cells were mounted with DAPI-containing media (Invitrogen). Images were taken by a fluorescent microscope (Eclipse E800; Nikon) and recorded using Openlab3 software (PerkinElmer).

Statistical Analysis Data were expressed as means �� SE. Statistical differences among the mean values of multiple groups were determined using analysis of variance followed by the Student��s t-test. P values < 0.05 were considered statistically significant. Results Lack of Nogo-B Reduces Fibrosis and Facilitates Apoptosis in Fibrotic Areas of the Mouse Cirrhotic Liver Sirius Red staining was performed in cirrhotic livers isolated from WT and Nogo-B KO mice that underwent CCl4 inhalation for 12 weeks Drug_discovery (Figure 1A).