In contrast, there was almost no difference in Bax or cytochrome distribution in hFis1 RNAi cells with or without apoptosis induction
In contrast, there was almost no difference in Bax or cytochrome distribution in hFis1 RNAi cells with or without apoptosis induction. hFis1. We also demonstrate in this study that mitochondrial fragmentation per se does not result in apoptosis. However, we provide further evidence that multiple components of the mitochondrial morphogenesis machinery can positively and negatively regulate apoptosis. INTRODUCTION Mitochondria are morphologically dynamic organelles that constantly divide and fuse to form small individual models or interconnected networks within the cell (Bereiter-Hahn, 1990 ). They reach an equilibrium between these two states in healthy cells by regulating the relative rates of organelle fusion and fission (Nunnari in yeast) (Otsuga in mitochondrial fission (Otsuga is usually involved in mitochondrial fission (Mozdy in yeast) (Olichon release (Karbowski (1:800; BD Biosciences PharMingen), or mouse monoclonal anti-DLP1/Drp1 (1:100; BD Transduction Laboratories, Lexington, KY) overnight at 4C trans-trans-Muconic acid followed by staining with goat anti-rabbit Alexa Fluor 594 (1:600; Molecular Probes, Eugene, OR) or goat anti-mouse Alexa Fluor 488 antibodies (1:600; Molecular Probes) for 2 h at RT. After washing, cells were mounted with SlowFade light antifade reagent (Molecular Probes) and analyzed by confocal microscopy. To visualize trans-trans-Muconic acid the mitochondria in living cells, 50 nM Mitotracker CMXRos (Molecular Probes) was added and incubated for 30 min before confocal microscopy. To analyze the mitochondrial membrane potential, cells were incubated for 20 min with 5 g/ml JC1 (Molecular Probes) in culture medium and observed by confocal microscopy. Images were captured with an LSM 510 Zeiss confocal microscope. Matrix-targeted photoactivable green fluorescent protein (mito-PAGFP)Cbased mitochondrial fusion assay was performed as explained previously (Karbowski for 5 min at 4C. Rabbit polyclonal to FANCD2.FANCD2 Required for maintenance of chromosomal stability.Promotes accurate and efficient pairing of homologs during meiosis. The supernatants (cytosolic fractions; S) were saved, and the pellets were solubilized in the same volume of mitochondrial lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40, 100 M PMSF, 10 g/ml leupeptin, 2 g/ml aprotinin), followed by centrifugation at 10,000 for 10 min at 4C, and the supernatants were used as heavy membrane (HM) fractions. Total cell lysates were prepared by solubilizing whole cells in Laemmli sample buffer and boiling them for 10 min. An aliquot of trans-trans-Muconic acid each sample was taken to determine protein concentrations by BCA protein assay kit (Pierce Chemical, Rockford, IL). Equivalent amounts of samples were run on 4C12% polyacrylamide gradient gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA), and immunoblotted. The primary antibodies, their dilutions, and their sources are as follows: anti-Fis1 (1:500; Axxora, San Diego, CA), anti-Opa1 (Zhu (1:1000; BD Biosciences PharMingen), anti-PARP (1:1000; BIOMOL Research Laboratories, Plymouth Getting together with, PA), and anti-actin (1:1000; Sigma-Aldrich, St. Louis, MO). Horseradish peroxidase-conjugated secondary antibodies (1:10,000) were used. Blots were detected by ECL Plus (Amersham Biosciences, Piscataway, NJ). Assessment of Apoptotic Cell Death Cells produced in chamber slides were treated as mentioned in the text or physique legends. Nuclei were stained with Hoechst 33342 (Molecular Probes) (1 g/ml; 15 min at RT) and visualized under the fluorescent microscope (for UV excitation), and cells were scored as normal or apoptotic nuclei in several fields. At least 200 cells altogether in each treatment were counted and are trans-trans-Muconic acid shown as a percentage of cells with apoptotic nuclei among total cells counted. Cells produced in a 10-cm culture dish were treated as mentioned above, harvested, and washed once with phosphate-buffered saline, and total DNA was isolated as explained previously (Lee and Shacter, 1997 ). After digestion with proteinase K and RNase A, the DNA was separated in 2% agarose gels and visualized with ethidium bromide under UV light. PARP is usually a well known caspase-3 substrate. During apoptosis, caspase-3 is usually activated, so PARP cleavage can be used as a marker of apoptosis. Cells produced in a 10-cm culture dish were treated.