MGP induces apoptotic death in activated HSCs.

<p>(<b>A</b>) Cytotoxic effect of MGP. Activated HSCs were treated with various concentrations of MGP (100, 200, and 400 µg/ml) for 24, 48, and 72 h. Viable cells were measured by the trypan blue dye exclusion method. (<b>B</b>) Morphological changes. HSCs and hepatocytes were incubated with MGP (400 µg/ml) for 72 h. Cell morphology was investigated under an Olympus IX70 phase-contrast microscope. (<b>C</b>) Induction of apoptosis by MGP. The HSCs were maintained in the presence or absence of MGP (400 µg/ml) for 48 h. After treatment, the TUNEL assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053988#s2" target="_blank">Materials and Methods</a>. The morphology of HSCs was again investigated under a phase-contrast microscope. TUNEL positive cells were considered apoptotic cells. The original magnification was 200×. (<b>D</b>) Activation of caspases by MGP. Activated HSCs were treated without or with MGP (400 µg/ml) for 48 h and the activity of each caspase was then assessed. (<b>E</b>) Inhibitors of caspases protected against MGP-induced apoptosis. Activated HSCs were pretreated with 100 µg/ml caspase inhibitor for 1 h and then treated with MGP (400 µg/ml) for another 72 h. After treatment, apoptotic cell count was assessed by the TUNEL assay. Significance for <i>p</i>-value<0.0001 is marked with ***. (<b>F</b>) Regulation of anti-apoptotic and proapoptotic molecules by MGP. Activated HSCs were treated without or with MGP (400 µg/ml) for 48 h. The levels of Bcl-2, Mcl-1, and Bax were determined by Western blot analysis.