Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was carried out for VEGF, VEGFR1, VEGFR2, and Col1a1 with Assays-on-Demand (Applied Biosystems). Western blotting of α-SMA on liver protein extracts was performed as described. 20 GAPDH was used as a loading control. Flow cytometric analysis of Ifn-γ in the cytoplasm of T cells was performed as described. 7 Isolation and culture of primary liver cells from CCl4-treated and untreated mice was performed as described by Taura et al. 22 The SV40-transformed mouse endothelial Wnt pathway cell line (SVEC) and the stellate cell line GRX 20 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with

4.5 g/L glucose (PAA Laboratories) and 10% heat-inactivated fetal calf serum (FCS). For chemokine stimulation, cells were starved click here in DMEM

containing 0.5% FCS (starving medium) for 16 hours and stimulated with recombinant mouse VEGF164 (20 ng, Biomol) in the presence or absence of recombinant mouse Cxcl9 (100 ng, Biomol) for 10 minutes. Western blots of phosphorylated and total VEGFR2 (KDR, kinase insert domain-containing receptor), PLCγ (phospholipase Cγ), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) (all antibodies from Cell Signaling Technology) were performed. The chemotaxis of endothelial cells to VEGF and its repression by Cxcl9 was assessed in a modified Boyden chamber system. Endothelial cells (1 × 104) were placed in the upper compartment C-X-C chemokine receptor type 7 (CXCR-7) in starving medium and were exposed to recombinant mouse VEGF164 (20 ng, Biomol) alone or in combination with recombinant mouse Cxcl9 (100 ng) in the lower compartment. After 4 hours of incubation, cell migration was analyzed by counting

cells of three random high-power fields (×100 magnification). All experiments were performed in quadruplicate. For quantification of endothelial and stellate cell proliferation a chemiluminescent immunoassay based on the measurement of bromodeoxyuridine (BrdU) incorporation during DNA synthesis was used (Cell Proliferation ELISA, BRDU, Roche Applied Science). 20 Briefly, cells (0.5 × 104) were cultured in starving medium for 16 hours and stimulated for 24 hours with VEGF and costimulated with Cxcl9 as mentioned. After BrdU labeling, fixation, and DNA denaturation, the BrdU incorporation was quantified by measuring the subsequent substrate reaction. For studying sinusoidal endothelial cell / hepatic stellate cell interactions in response to Cxcl9, we performed experiments with conditioned medium. Cultured endothelial cells were stimulated with or without VEGF ± Cxcl9 (see above) for 24 hours and the supernatant was harvested for cell migration and proliferation experiments of stellate cells. The composite of endothelial cell migration and proliferation were performed in a scratch assay.