As this occurred in several repeated experiments, the hepatocytes were not investigated further. The movement of procaspase-9 into buy Ganetespib and out of the nuclei appeared to occur in an organized manner. Whether this transport depended on the assembly of microtubules was tested by the microtubule-disrupting agent vinblastine. Ten μM vinblastine was added to the cell culture medium 4 hours after the isolation of hepatocytes (Fig. 3B). Its addition prevented the transport of procaspase-9
into cell nuclei. Therefore, procaspase-9 is transported along the microtubules from the cytoplasm to the nuclei. The mitochondrial morphology changed over time in cultured hepatocytes (Fig. 4A). The same types of changes were observed when the mitochondria were labeled by a MitoTracker, fluorescently labeled Tim23 (an integral mitochondrial inner membrane protein) and by fluorescently labeled streptavidin, which specifically labels mitochondria by binding
to biotinylated proteins of mitochondrial matrix.21 Mitochondria were labeled by streptavidin in this study because the strengths of streptavidin fluorescent signals did not vary during the incubation time of primary hepatocytes. Selleck Compound Library Mitochondria appeared circular in liver slices and in freshly isolated cells for up to 8 hours. They appeared dispersed after 24 hours postisolation (Fig. 4A), whereas mitochondria formed longer tubules after 3 days in culture. It seemed that mitochondrial fission predominated immediately after the isolation of primary hepatocytes. As in the case of the nuclear shift of procaspase-9, the fission of mitochondria was reversible too. Despite the
changes in mitochondrial morphology, there was neither Cyt-c leakage from dispersed mitochondria nor was there a decrease in MMP (Fig. 4B). The ratio between the potentials of the energized mitochondria and when MMP was dissipated by FCCP was statistically higher at 1 day of hepatocyte culture compared to immediately after isolation (P = 3 × 10−7, unpaired two-tailed Student’s t test). The difference between the MMPs of hepatocytes immediately after isolation and selleck kinase inhibitor those cultured for 1 day is relatively small and could be due to the presence of some cells that were damaged during isolation in the sample that was assayed immediately thereafter. The change in cellular location of Bax may be another feature of early apoptosis. We localized Bax to the cytoplasm of hepatocytes in rat liver sections (Fig. 5A). In contrast, only minor amounts of it were cytoplasmic, whereas most of it was in the nuclei of the primary hepatocytes cultured for 24 hours. Bax remained predominantly in the nuclei throughout the culturing of primary hepatocytes; it shifted to cytosol and mitochondria whenever apoptosis was induced by STS. The antibody used for labeling of Bax detected a single band of 22 kDa (Fig. 5B). This proves that Bax was labeled specifically.