7B). Hepatocytes expressed Insig-2 protein, whereas we could not observe any expression of Insig-2 in HSCs (Fig. 7B). A Scap trypsin cleavage assay[13] was subsequently performed to examine whether or not cholesterol-induced Scap conformational changes occurred in these cells. Scap, without cholesterol-induced conformational changes, yields a protected band of 27 kDa on sodium dodecyl sulfate-polyacrylamide Y-27632 price gel electrophoresis (SDS-PAGE), whereas Scap, with the conformational change, yields a protected band of 26 kDa. Our data showed that the cholesterol-induced Scap conformational change in activated HSCs occurred to the same degree as that in quiescent HSCs or hepatocytes (Supporting

Fig. selleck inhibitor 10A,B). LDL treatment decreased the nuclear level of SREBP2 in quiescent HSCs. Treatment with Scap-siRNA or Insig-2-overexpression vector enhanced the effect, whereas treatment with Insig-1-siRNA counteracted the effect (Fig. 7C, upper and middle). However, LDL treatment did not affect the nuclear level of SREBP2 in activated HSCs; overexpression of

Insig-1 or Insig-2 in HSCs significantly decreased the nuclear level of SREBP2 after the addition of LDL (Fig. 7C, lower). LDL treatment increased the level of the Scap-Insig-1 complex in quiescent HSCs, whereas cotreatment with Scap-siRNA or Insig-1-siRNA reversed this change (Fig. 7D). We could not detect any Scap-Insig-2 complex Rebamipide in quiescent HSCs after the addition of LDL. Overexpression

of Insig-2 increased the level of the Scap-Insig-2 complex in LDL-treated quiescent HSCs (Fig. 7D). On the other hand, neither the Scap-Insig-1 nor the Scap-Insig-2 complex could be detected in activated HSCs treated with LDL or not (Fig. 7E). Overexpression of Insig-1 increased the level of the Scap-Insig-1 complex in activated HSCs treated with LDL, and similarly, overexpression of Insig-2 increased the level of the Scap-Insig-2 complex after treatment with LDL (Fig. 7E). In addition, the feedback regulation system of cholesterol homeostasis impacted the sensitization of HSCs to TGFβ-induced activation, in a manner similar to the FC accumulation system mediated by LDLR or miR33a (Supporting Fig. 11). The Insig-1 expression level was significantly lower in HSCs from the MCD and HF diet-fed groups than in those from the corresponding control diet-fed groups (Fig. 8A,B; Supporting Fig. 12A,B). These decreases were significantly enhanced by the increased intake of cholesterol (Fig. 8A,B; Supporting Fig. 12A,B). We could not detect any difference in the Scap expression level in HSCs among the groups (Fig. 8A,B; Supporting Fig. 12A,B). Furthermore, Insig-1 protein was abundant in quiescent HSCs but its level declined at days 3 and 5, and day 7 HSCs (Supporting Fig. 12C). We could not detect any significant difference in the Scap expression level among the groups (Supporting Fig. 12C).