The baseline selleck chemicals level of caspase-6 expression
was reduced to 0.75 ± 0.036 au in PV-MITO-GFP cells in comparison with the control (P < 0.001), and it increased to 1.98 ± 0.09 au in nontransfected cells (0.97 ± 0.03 au in PV-MITO-GFP cells, P < 0.001, n = 3; Fig. 4D). Conversely, the expression of genes encoding antiapoptotic proteins was up-regulated after Ca buffering (Fig. 4E-G). Bcl-2 gene expression increased to 1.21 ± 0.13 au in PV-MITO-GFP cells in comparison with the control (P < 0.001, n = 3) and remained higher upon STA treatment (1.19 ± 0.17 versus 0.63 ± 0.09 au in the control, P < 0.001, n = 3; Fig. 4E). Similarly, the expression of mcl-1 and bcl-xL genes increased to 1.2 ± 0.06 and 1.41 ± 0.10 au, respectively, in PV-MITO-GFP cells and to 1.0 ± 0.05 and 1.0 ± 0.06 au, respectively, in the control (P < 0.001, n = 3). After the STA treatment, the expression
levels of mcl-1 and bcl-xL remained high (1.18 ± 0.06 and 1.26 ± 0.10 au, respectively) in PV-MITO-GFP cells (0.46 ± 0.02 and APO866 nmr 0.73 ± 0.06 au in the control, P < 0.001, n = 3; Fig. 4F,G). To examine whether the expression of these genes was also altered at the protein level in SKHep1 cells expressing PV-MITO-GFP, we performed immunoblotting for the antiapoptotic protein bcl-2 and the proapoptotic protein bax (Fig. 4H-J). The expression of the bcl-2 protein increased to 1.15 ± 0.09 au in cells expressing PV-MITO-GFP compared with 0.56 ± 0.08 au in control cells (P < 0.001, n = 3), whereas the expression of the bax protein decreased to 0.84 ± 0.09 au in cells expressing PV-MITO-GFP compared with 1.14 ± 0.09 au in control cells (P < 0.05, n = 3). Similar results were observed in cells treated with STA. These Rebamipide findings suggest that Ca buffering directs the expression ratio of proapoptotic and antiapoptotic protein members toward a predominantly antiapoptotic pathway. For the determination of whether the decrease in cell death observed in PV-MITO-GFP cells was associated with changes in proliferation, SKHep1 cells were synchronized in G0 by serum withdrawal, transfected with
the target constructs, and assayed for BrdU incorporation. No increase in cell proliferation was observed in PV-MITO-GFP cells in comparison with control cells or cells expressing MITO-GFP (supporting Fig. 1). However, with agonist-induced cell death, BrdU uptake was lower in cells expressing MITO-GFP versus cells expressing PV in the mitochondria (51.1% ± 5.3% for MITO-GFP versus 79.4% ± 3.6% for PV-MITO-GFP, P < 0.001, n = 3). Together, these results suggest that Ca buffering preferentially prevents cells from undergoing apoptosis instead of stimulating proliferation. Liver regeneration requires both increased cell proliferation and reduced apoptosis.25 The role of Ca signaling in apoptosis is well known, but its role in liver regeneration has not been studied. Therefore, we investigated the involvement of Ca in liver growth after two-thirds hepatectomy (i.e., PH).