Supplementary material for this article can be found on the H EPATOLOGY website ( ).

jws-hep.21427.fig1.pdf83K Supplementary Fig. 1 . Scheme of the co-culture model. After liver perfusion and elutriation, 450,000 KC were plated on the cell culture inserts in 4 mL of DMEM/F12 containing 10% FBS. HSC were seeded on the bottom either on polystyrene plates or on plates coated with Matrigel at a density of 150,000 cells in 4 mL of DMEM/F12 with 10% FBS. One day later, the medium was discarded and, either empty inserts (co-culture controls) or inserts containing KCs were transferred onto the HSCs. Fresh medium (4 mL) without serum were added and samples of HSC lysates or culture medium were collected at selected time points according to the experiment.
jws-hep.21427.fig2.pdf114K Supplementary Fig. 2 . Phenotypic changes of HSCs in co-culture with KCs on regular polystyrene plates (left panels) or on Matrigel-coated plates (right panels) in the absence of serum. At each time-point, the inserts containing the KC were removed and pictures of HSC were taken (light micrographs). HSC co-cultured with KC under both conditions showed phenotypic changes indicative of higher activation than those of HSC cultured alone. Co-cultured HSC plated on Matrigel began to extend cellular processes or filopodia and displayed spindle-like shape with distal fine branches forming polygonal structures (Magnification = 200?).
jws-hep.21427.fig3.pdf40K Supplementary Fig. 3 . The expression of -sma in the co-cultures was assessed by Western blot analysis using 5 &&num;956;g of cell lysate protein from HSCs cultured alone or with KCs in the absence of serum plated either on polystyrene plates or on Matrigel-coated plates. A representative Western blot analysis is depicted with the densitometry analysis under the blot. Results were compared to those of HSCs grown alone at 1 day for cells plated on polystyrene dishes and to those of the co-culture at 6 days for cells grown on Matrigel-coated plates due to lack of signal in the control. n = 3, *P < .05, **P < .01, and ***P < .001 for HSCs/KCs versus HSCs either on polystyrene plates or on Matrigel.

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