kNF kB action was determined using Trans-am package from Active Motif based on the manufacturers directions. Polyvinyl pyrrolidone free polycarbonate membranes with 8 mm pores, which split up the upper and lower wells in a transwell chamber program, were covered with type IV collagen on the upper side and type I collagen on the pifithrin a lower side, as previously described. The bottom wells of the chamber were stuffed with DMEM, and 26104 cells/ well, which had been serum starved for 24 h, were added in to the upper chamber. HMGB1 was added into the upper chamber like a direct haptotactic stimulant, and into the lower chamber being an indirect chemotactic stimulant, to mimic the in vivo autocrine and paracrine mechanisms of cytokines respectively. The chamber was incubated at 37uC for 4 h allowing the migration of cells through the membrane into the lower chamber. The moved cells were mentioned in six random fields on the phase contrast microscope and stained with Hema3 according to the suppliers Cellular differentiation protocol. HSCs were organized with RIPA buffer containing protease inhibitor combination and washed twice with ice cold PBS. The samples were separated by SDS PAGE and then transferred onto a polyvinylidene difluoride membrane using SemiDry Transfer Cell. The polyvinylidene difluoride membrane was blocked with 5% non fat milk for 3 h accompanied by incubation with primary antibody in TBST overnight at 4uC with gentle shaking, the specific primary antibodies against JNK, p JNK, PI3K, p PI3K, Akt, p Akt, NF kB, IkB and p IkB. The blots were incubated with the HRP conjugated anti GAPDH antibody for 1 h at room temperature. The ratio of each protein to GAPDH was calculated because the relative quantification. First HSCs, which had been incubated with human TLR4 neutralizing antibody for 1 h, were collected and added into the upper chamber of modified transwell chamber program, and then HMGB1 was added into the upper chamber as a strong haptotactic stimulant or into the low chamber as an indirect chemotactic stimulant to test if the TLR4 ubiquitin-conjugating is involved in HMGB1 induced HSCs migration. Second, TLR4 neutralizing antibody was incubated with human principal HSCs for 1 h, and then HMGB1 was included into the culture medium to determine whether the TLR4 is involved in HMGB1 induced HSCs expansion and activation of JNK, PI3K/Akt and NF kB. Next, JNK inhibitor and PI3K inhibitor were incubated with human key HSCs for 1 h, and then HMGB1 was added to the culture medium to find out if the JNK and PI3K/Akt signal pathways are involved in HMGB1 induced HSCs expansion and pro fibrotic effects. Finally, HSCs, which had been incubated with SP600125 and LY 294002 at above concentrations for 1 h, were then collected and added into the upper chamber of revised transwell chamber program and HMGB1 was added into the upper chamber or the reduced chamber to try perhaps the JNK and PI3K/Akt transmission pathways are involved in HMGB1 induced HSCs migration.