1. Mitochondrial HMGB1 is linked to mitochondrial functions Western blot with separated cellular components from the cerebellar tissues of the three genotypes of mice showed that HMGB1 was decreased not only in the nucleus but also in the cytosol and mitochondria of mutant Atxn1‐KI mice.
Mitochondrial HMGB1 is linked to mitochondrial functions Western blot with separated cellular components from the cerebellar tissues of the three genotypes of mice showed that HMGB1 was decreased not only in the nucleus but also in the cytosol and mitochondria of mutant Atxn1‐KI mice. The downregulation in the three subcellular fractions was reversed in the double‐transgenic mice. The band intensity was quantified and is shown in the lower graphs. The data are presented as mean ± SD. Statistical analysis involved Student's t‐test and one‐way ANOVA followed by post hoc Tukey's HSD (honestly significant difference) test. Immunoelectron microscopy of the cerebellar cortex of control mice, with staining using an anti‐HMGB1 antibody. Nucleus‐dominant distribution of the gold–silver complex particles was found (arrows in the upper left panel), although the particles were also found in the cytoplasm. In the cytoplasm (lower left panel), the mitochondrial membrane (arrow) and matrix (arrowhead) were stained. Higher‐magnification images of the mitochondrial matrix staining are shown in the upper and lower right panels. Western blot detection of HMGB1 in mitochondria purified by multiple methods. Lane 1: the mitochondrial fraction purified from the brain by the Percoll density gradient centrifugation method (Sims & Anderson, 2008), Lane 2: the mitochondrial fraction purified from the mouse liver by the other centrifugation method (Shimizu et al, 1998), Lane 3: mitochondrial fraction purified from the mouse brain using a commercial isolation kit), Lane 4: the cytosol fraction from the mouse brain isolated using the Mitochondria Isolation Kit (Thermo Scientific, IL, USA) and Lane 5: a nuclear fraction from the mouse brain isolated using the same kit. All the methods revealed the presence of HMGB1 in mitochondria. Proteinase K was added to the mitochondrial fraction prepared by the Percoll density gradient centrifugation method, to exclude the possibility of contamination with the nuclear or cytosolic fraction. Addition of proteinase K before membrane perforation did not affect HMGB1 dramatically decreased HMGB1 after membrane perforation, indicating that HMGB1 is located in the mitochondria. Other mitochondrial proteins including Cox IV and cytochrome c at the inner mitochondrial membrane or TFAM that binds to mitochondrial DNA inside the inner mitochondrial membrane showed a similar pattern of changes, whereas Tom20 at the outer mitochondrial membrane was digested before membrane perforation. Source data are available online for this figure.
Hikaru Ito et al. EMBO Mol Med. 2014;emmm
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