MF-HSCs were treated with 1 ��mol/L STS for 0, 2, 4, 6, 8, and 10 hours. A: Representative Western blot analysis of apoptotic markers, including cleaved PARP; cleaved … Lack of Nogo-B Does Not Influence Hepatocyte Apoptosis in Response to STS To investigate the possibility that Nogo-B may modulate selleckchem apoptosis in hepatocytes, hepatocytes from WT and Nogo-B KO mice were treated with STS (Figure 4). Hepatocytes required a higher dose of STS than MF-HSCs to induce apoptosis (10 versus 1 ��mol/L, respectively). Moreover, there was no difference in the levels of apoptotic markers between WT and Nogo-B KO hepatocytes. These results indicate that the absence of Nogo-B preferentially sensitizes MF-HSCs rather than hepatocytes to apoptosis under conditions of experimental cirrhosis.

Figure 4 Lack of Nogo-B does not influence hepatocyte apoptosis in response to STS. Hepatocytes were isolated from WT and Nogo-B KO mice and treated with 10 ��mol/L STS for 0, 2, 4, 6, 8, and 10 hours. Heat shock protein 90 (Hsp90) was used as a loading … Knockdown of Nogo-B Increases Apoptosis in Human HSCs (LX2) To confirm the role of Nogo-B in apoptosis in human MF-HSCs, LX2 cells were transfected with Nogo-B small-interfering RNA to suppress Nogo-B expression. We then tested apoptosis in those cells treated with 100 nmol/L STS for 0, 2, 4, 6, 8, and 10 hours (Figure 5). Nogo-B small-interfering RNA resulted in a 66% reduction of Nogo-B expression. Similar to the results in mouse MF-HSCs, the levels of cleaved PARP and caspase-3 were increased in LX2 cells treated with Nogo-B small-interfering RNA compared with controls, however, the levels of cleaved caspase-9 and Bcl-xL did not differ.

Consistent with mouse MF-HSCs, knockdown of Nogo-B resulted in enhanced apoptosis in human MF-HSCs in response to STS. Figure 5 Knockdown of Nogo-B increases apoptosis in human hepatic stellate cells (LX2). LX2 cells with or without Nogo-B siRNA were treated with 100 nmol/L STS for 0, 2, 4, 6, 8, and 10 hours. Western blot analysis for apoptotic markers, including cleaved PARP, … Overexpression of Nogo-B Blocks Apoptosis in Human HSCs (LX2) To determine the effect of Nogo-B overexpression on apoptosis of human MF-HSCs, LX2 cells were transfected with HA-tagged human Nogo-B plasmid and treated with 100 nmol/L STS for 8 hours.

The transfection resulted in a sevenfold higher level of Nogo-B over the endogenous Nogo-B level found in nontransfected LX2 cells (data not shown). As shown in Figure Dacomitinib 6, the majority of HA�CNogo-B�Cpositive cells were negative for cleaved caspase-3. In fact, the percentage of cleaved caspase-3�Cpositive cells was significantly lower in HA�CNogo-B�Cpositive cells than in nontransfected cells (4.2% versus 29.4%, respectively; P < 0.01). These results suggest that MF-HSCs become resistant to apoptosis with Nogo-B overexpression. These data also lend support to our earlier data that the lack of Nogo-B facilitates apoptosis of MF-HSCs.