fig8

Figure 8. Primary cultures of HSCs isolated from L-SACC1 and wild-type mice. HSCs were isolated as described in Methods and cultured in DMEM with 450 mg/dL glucose, 10% FCS, and 10% HS. Cells were harvested at times indicated on individual panels for Western blot analysis and analyzed by Alamar blue assay for proliferation on day 6. (A) shows Western blot analysis demonstrating increased production of COL1 and α5 integrin by HSCs isolated from L-SACC1 mice compared to cells from wild-type animals; (B) represents a Western blot analysis on days 3, 5, and 7, showing significantly higher COL1 levels in L-SACC1 than in wild-type mice only on day 7. This suggests that HSCs from L-SACC1 mice do not undergo activation earlier in culture. In contrast, α5 integrin levels are increased even after 3 days in culture, with a more pronounced increase in HSCs from L-SACC1 compared to those from wild-type mice; (C) shows no difference in HSC proliferation in primary culture between L-SACC1 and wild-type cells. Cell proliferation was determined by a colorimetric change in the medium measured as a difference in culture media absorbance at 570 and 600 nm. HSCs: Hepatic stellate cells; DMEM: Dulbecco’s modified Eagle Medium; COL1: type I collagen.