![]() Lactate dehydrogenase (LDH) expression was checked as a cytosolic marker and Hsp60 expression was assessed as a mitochondrial membrane marker. c) Endogenous EndoG release was assessed by Western Blot in cytosolic extracts from control cardiomyocytes (scr) and cardiomyocytes expressing low levels of Bnip3 (achieved by shRNA-driven knockdown) in standard and ischemic conditions. The graph shows the percentage of cardiomyocytes with diffuse pattern of the EndoG-FLAG staining in control and ischemic cultures of scrambled-transduced cells (scr) and cells transduced with viruses for Bnip3 silencing (Bnip3), counted in three independent experiments. b) Quantification of the experiments described in a). Similar results were obtained in three independent experiments. Arrowheads: ischemic cardiomyocytes where EndoG-FLAG expression excludes the nucleus. A merged image including Hoechst nuclear staining is included for ischemic cardiomyocytes to localize the nuclei. Arrows: cardiomyocytes where EndoG-FLAG expression includes the nucleus. Bnip3i: cardiomyocytes with silenced expression of Bnip3 by shRNA. Error bars are s.e.m.īNIP3 controls EndoG release from cardiac mitochondria during ischemia.Ī) Immunofluorescence of EndoG-FLAG in overexpressing neonatal cardiomyocytes cultured in control (control) and ischemic (ischemia) conditions for 12 hours. total nuclei (blue+pink) from three independent experiments counted in duplicates. TUNEL-positive nuclei (pink) are expressed as mean vs. Gel images are representative of three independent experiments. TUNEL staining was detected as a red fluorescent signal, which, in combination with blue Hoechst nuclear staining produced a pink hue. d) DNA integrity was measured by the TUNEL assay following the same treatments as in B. LMWF: Low molecular weight DNA fragmentation detected by conventional agarose gel electrophoresis from the same cell extracts. HMWF: High molecular weight DNA fragmentation detected by pulse-field electrophoresis as reported in the Materials and Methods section. A sample from scrambled-transduced cardiomyocytes treated with ischemia in the presence of 100 µM pan-caspase inhibitor z-VAD-fmk was added to show that DNA fragmentation was caspase-independent in this paradigm. Cardiomyocytes were transduced 5 days before ischemia with constructs for the silencing of Bnip3, Nix or EndoG genes. c) Agarose gel electrophoresis of DNA extracts of cardiomyocytes cultured in normal conditions or after 16 hours of experimental ischemia. b) Efficiency of EndoG shRNA-mediated knock down and specificity of the new anti-EndoG antibody was analyzed by Western blot in the same conditions than in a. A representative image is shown from three independent experiments.īnip3 and EndoG trigger caspase-independent TUNEL positive DNA fragmentation in ischemic cardiomyocytes.Ī) Efficiency of Bnip3 and Nix shRNA-mediated knock down was analyzed by RT-PCR and / or Western blot from postnatal cardiomyocyte total RNA or protein extracted at day 4 post-transduction. Primary heart fibroblasts and neonatal liver samples were added for comparison. c) Neonatal cardiomyocytes were treated with ischemia and the abundance of EndoG, Bnip3 and proteins involved in the caspase-dependent pathway was analyzed by Western blot in extracts obtained at different time points. A representative image is shown from three independent experiments. b) Western blot detection of EndoG, Bnip3 and proteins involved in caspase-dependent cell death in total protein extracts of ventricles from rats of different ages ranging from embryonic day 20 to adulthood. Detection of EndoG and Bnip3 was performed in two different sets of independent samples with similar results. Both proteins are expressed mainly in the heart and skeletal muscle. EndoG is detected as a single band (∼27 kDa) and Bnip3 is detected as a group of bands around the 26 kDa mark (asterisk), in agreement with the information provided by the manufacturer. EndoG and Bnip3 are most highly expressed in rat heart, and are located to cardiac myocytes thorough development and during ischemia.Ī) Western blot analysis of EndoG, using a new anti-EndoG antibody (AntibodyBcn, BCN4778) and Bnip3 protein expression in total protein extracts from different adult rat tissues. ![]()
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