Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Diagram of coronal rat brain section showing the site of injury and photographed region (4 A). Representative immunofluorescence images (10 ×) of ipsilateral cortex GFAP, ED-1, and HMGB1 immunoreactivity (green) combined with DAPI (blue) at 14 days post-TBI (10 days post-alcohol exposure) (4 B).

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Immunofluorescence

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Quantification of ipsilateral cortex GFAP, ED-1, and HMGB1 immunoreactivity at 14 days post-TBI (10 days post-alcohol exposure). Images are quantified as % area of positive staining in 1.035 mm2 (10×). Values are shown as means ± SEM. For GFAP and ED-1, Sham/Air n = 4, Sham/Alcohol n = 4, TBI/Air n = 4, TBI/Alcohol n = 6. For HMGB1, Sham/Air n = 4, Sham/Alcohol n = 5, TBI/Air n = 5, TBI/Alcohol n = 7 (each n represents the average of 3 images taken from each animal) * p < 0.05 of the TBI groups vs. time-matched sham controls; $ p < 0.05 of the TBI/Alcohol group vs. TBI/Air group, by two-way ANOVA (5 A, 5 C, and 5 E).

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Staining

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Primary and Secondary Antibodies.

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Differential location of HMGB1 expression in HDFs and KFs. ( a ) HMGB1 expression levels by immunofluorescence. HMGB1 expression (red) was mainly detected in the HDF nuclei. ( b ) HMGB1 expression was observed in KFs, but was more abundant in the cytosol and extracellular space. ( c and d ) HMGB1 protein expression. The levels of proteins were assessed by western blot. α-tubulin was used as a loading control for cytosol; histone was as a nuclei protein control. Relative levels of HMGB1 in the cytoplasmic fraction were significantly elevated in the KFs. ( e and f ) Stimulation of HDFs with LPS (100 ng/mL) for 24 h and 48 h resulted in increased time-dependent expression of HMGB1 protein in the cell lysate (** p < 0.01). ( g ) Whole cell lysates were fractionated and the levels of HMGB1 in the cytoplasmic and nuclear fractions were determined by western blotting to determine whether LPS treatment induced HMGB1 translocation. HMGB1 translocated from nucleus to cytoplasm in HDFs under LPS treatment (500 ng). Dermal fibroblasts cell line was purchased as primary cell line from the ATCC (American Type Culture Collection, Manassas, VA, USA). Primary keloid fibroblast cell line were obtained from human keloid explant under IRB protocol. Both cells were used for experiment after 2~3 passage.

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Expressing, Immunofluorescence, Western Blot, Translocation Assay

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Role of HMGB1 as a profibrotic molecule. ( a ) An MTT assay revealed a significant increase in time-dependent proliferation activity in the HMGB1-treated HDFs (50 and 100 ng; ** p < 0.01). ( b ) Type I collagen mRNA expression was equivalently increased in the HDFs treated with HMGB1 (100 ng) or TGF-β1 (10 ng) (* p < 0.05, ** p < 0.01). There was also an additive increase of type I collagen mRNA expression after simultaneous treatment with HMGB1 and TGF-β1 ( § p < 0.05). ( c ) Levels of collagen types I and III were detected by western blotting in HDF lysates treated with HMGB1 (100 ng) for 24 and 48 h. ( D ) The protein expression levels of collagen types I and III increased significantly after 24 h (** p < 0.01). ( e) The protein expression level of α-SMA was assessed by western blotting in HDF cells treated with HMGB1 (50 and 100 ng). ( f ) Stimulation of HDFs with HMGB1 resulted in increased protein expression of α-SMA (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: MTT Assay, Activity Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: HMGB1 induced TGF-β1, Smads, Erk 1/2, Akt, and NF-κb in HDFs. ( a – c ) Effects of HMGB1 on TGF-β1 signaling pathway in vitro . Significant increases in the expression levels of TGF-β1, Smad 2, and Smad 3 mRNA were observed in the HMGB1 (50 ng)-treated HDFs (** p < 0.01). ( d ) Effects of HMGB1 on TGF-β and phosphor-Smad 2/3 complex protein expressions. ( e , f ) TGF-β and phosphor-Smad 2/3 complex protein levels were significantly increased in the HMGB1 (100 ng)-treated HDFs (** p < 0.01). ( g ) Western blotting analysis in normal dermal spheroids. Increased expression of TGF-β and phosphor-Smad 2/3 complex protein were observed in normal dermal spheroids treated with HMGB1 (400 ng and 1000 ng). ( h ) The levels of Erk 1/2, Akt, and NF-κb protein were detected by western blotting in HDFs lysates treated with HMGB1 (50 ng and 100 ng). ( i to k ) Erk 1/2, Akt, and NF-κb protein levels in HMGB1 (100 ng)-treated HDFs were significantly increased by 1.5-, 2.0-, and 2.3-fold, respectively, versus non-treated HDFs (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: In Vitro, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: HMGB1 reduced MMP 1 and TIMP-1 mRNA expression. HDFs were treated with 50 ng/mL of HMGB1 for various times, and examined using real-time RT-PCR. ( a and c ) MMP-1 mRNA levels were significantly reduced, but TIMP1 mRNA level was increased in 48 hr after treatment with HMGB1 (** p < 0.01). ( b ) MMP-2 mRNA expression increased up to 24 h, but the increase was not statistically significant.

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Effect of HMGB1 on normal dermal spheroids. ( a ) Picrosirius red staining of HMGB1-treated normal dermal spheroids. Collagen deposition increased. The HMGB1 dose dependency of dense and coarse collagen bundles was investigated. ( b ) Semi-quantitative analysis revealed that treatment with 400 and 1000 ng of HMGB1 significantly increased collagen deposition in normal dermal spheroids by 2.9- and 2-fold, respectively, versus non-treated dermal spheroids (** p < 0.01). ( c ) Western blotting analysis of HMGB1-treated normal dermal spheroids for the detection of type-I and -III collagen protein expression level ( d ) Immunohistochemical staining of HMGB1-treated normal spheroid sections for the detection of types I and III collagen, elastin, and fibronectin protein. Increased expression of ECM components (collagen types I and III, elastin, and fibronectin protein) was examined versus nontreated spheroids. Original magnification: 400×. ( e to h ) Semi-quantitative image analysis of ECM protein expression. The expression levels of type I collagen, type III collagen, elastin, and fibronectin increased significantly versus nontreated spheroids (* p < 0.05; ** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Staining, Western Blot, Expressing, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Immunohistochemical analysis of HMGB1 and its receptors (RAGE and TLR4) in human keloid tissues. ( a ) Higher expression levels of HMGB1 and its receptors (RAGE and TLR4) were observed in the region of the keloid tissues than in the adjacent normal tissue. Original magnification: 400×. ( b to d ) The comparison of HMGB1, RAGE, and TLR4 expression levels between keloid tissues and extra-lesional normal tissue (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: A tactical nanomissile mobilizing antitumor immunity enables neoadjuvant chemo-immunotherapy to minimize postsurgical tumor metastasis and recurrence

doi: 10.1016/j.apsb.2022.09.017

Figure Lengend Snippet: Mit elicited the immunogenic death of B16–F10 cells and the preparation diagram of mitoxantrone@anti-PD-L1/azobenzene-lipo (MPAL). (A) CRT detections on B16–F10 cells after being treated with different concentrations of Mit using flow cytometry. (B) CRT expression on B16–F10 cells in response to Mit was observed by confocal microscopy. Scale bar = 20 μm. Release of (C) HMGB1 and (D) ATP by B16–F10 cells after Mit treatment. ( ∗∗∗ P < 0.001 indicates the statistical difference between each group and the group without Mit) (E) The expression of CD86 on BMDCs after co-cultured with Mit-treated B16–F10 cells. (F) The vaccination method was used to identify Mit-induced immunogenic B16–F10 cell death. (G) The synthesis routes of AZO. (H) Schematic illustration of the preparation steps of MPAL. Data are presented as mean ± SD ( n = 3). ∗∗∗ P < 0.001.

Article Snippet: For HMGB1 detection, cells were permeabilized, fixed, and then stained with FITC conjugated anti-mouse HMGB1 antibody (Bioss) before flow cytometric detection.

Techniques: Flow Cytometry, Expressing, Confocal Microscopy, Cell Culture

Journal: EMBO Molecular Medicine

Article Title: HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

doi: 10.15252/emmm.201404392

Figure Lengend Snippet: The expression of exogenous HMGB1-FLAG (detected with an anti-FLAG antibody) and endogenous+exogenous HMGB1 (detected with an anti-HMGB1 antibody) was tested by immunohistochemical analysis. Nuclear and cytoplasmic signals were reduced in Atxn1-KI mice but restored in double-transgenic mice. The mean ± SD are shown in the lower graph. Aggregate formation was tested by immunohistochemical analysis of cerebellar tissues of background C57BL/6 mice, mutant Atxn1-KI mice and double-transgenic mice. Ubiquitin-positive aggregates were observed in mutant Atxn1-KI mice and double-transgenic mice (upper panels); the ratio of aggregate-positive Purkinje cells did not differ between the four mutant Atxn1-KI mice and the four double-transgenic mice (lower graph). The data are presented as mean ± SD. Western blot analysis with anti-ubiquitin antibody confirmed that aggregate formation was similar in mutant Atxn1-KI mice and double-transgenic mice. The rotarod performance test revealed improvement in the motor activity of double-transgenic mice over that of mutant Atxn1-KI mice. The time on rotarod was the mean value of the times from day 1 to day 3. The symptomatic onset in mutant Atxn1-KI mice was 7 weeks; improvement in double-transgenic mice lasted from 9 to 21 weeks. The data are presented as mean ± SD. Statistical analysis was performed with the Bonferroni–Dunn test and Student's t -test. Details of the rotarod performance of the three genotypes of mice (WT, Atxn1-KI and double-transgenic) from day 1 to day 3 at 5, 9 and 21 weeks. The data are presented as mean ± SD. The survival rates of the four genotypes of mice. The mean and maximum lifespans of double-transgenic mice were nearly 30% longer than those of mutant Atxn1-KI mice. The 50% survival duration was extended from 217 days (Atxn1-KI) to 282 days (Atxn1-KI;HMGB1), and the maximum survival duration was extended from 274 days (Atxn1-KI) to 360 days (Atxn1-KI;HMGB1). The effect of HMGB1 on the lifespan was assessed by Kaplan–Meier analysis, and the end-point postponement was significant in the log-rank test ( P = 0.0025). Histological evaluation of the double-transgenic mice. Quantitative analysis of histological parameters in the three groups of mice showed that reduction in the molecular layer thickness was reversed in double-transgenic mice (upper graph; Student's t -test, P < 0.05). The mean thickness was quantified in more than 10 visual fields per mouse, and the mean ± SD was calculated for nine mice in each group. The number of Purkinje cells, similarly calculated, was not changed in mutant Atxn1-KI mice or in double-transgenic mice (lower graph). The data are presented as mean ± SD. * P < 0.05 in Student's t -test, # P < 0.05 in one-way ANOVA followed by post hoc Tukey's HSD (honestly significant difference) test. Source data are available online for this figure.

Article Snippet: These sections were incubated sequentially with 1% BSA for 20 min, with an anti-HMGB1 antibody (AHM0915, dilution 1:100; ATGen, Seongnam-si, Korea) overnight at 4°C and with mouse anti-IgG antibodies conjugated with 1.4-nm gold particles (#2002, dilution 1:50; Nanoprobes, NY, USA) for 1 h at room temperature.

Techniques: Expressing, Immunohistochemical staining, Transgenic Assay, Mutagenesis, Ubiquitin Proteomics, Western Blot, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

doi: 10.15252/emmm.201404392

Figure Lengend Snippet: 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, ), Lane 2: the mitochondrial fraction purified from the mouse liver by the other centrifugation method (Shimizu et al , ), 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.

Article Snippet: These sections were incubated sequentially with 1% BSA for 20 min, with an anti-HMGB1 antibody (AHM0915, dilution 1:100; ATGen, Seongnam-si, Korea) overnight at 4°C and with mouse anti-IgG antibodies conjugated with 1.4-nm gold particles (#2002, dilution 1:50; Nanoprobes, NY, USA) for 1 h at room temperature.

Techniques: Western Blot, Mutagenesis, Transgenic Assay, Immuno-Electron Microscopy, Control, Staining, Membrane, Purification, Gradient Centrifugation, Centrifugation, Isolation

Journal: EMBO Molecular Medicine

Article Title: HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

doi: 10.15252/emmm.201404392

Figure Lengend Snippet: The accumulation of MitoTracker Deep Red, a far red-fluorescent dye indicator of the mitochondrial membrane potential, in the mitochondria of mutant Atxn1(86Q)-DsRed-transfected HeLa cells was reduced by co-expression of HMGB1-GFP. A knock-down of HMGB1 by two types of HMGB1-siRNA (HMGB1_A, HMGB1_B) decreased the number of mitochondria with the normal membrane potential (stained red with JC-1) and increased the number of mitochondria with an abnormal membrane potential (stained green with JC-1). NC: negative control siRNA. Right panels show HMGB1 signals in the transfected cells. Expression levels of HMGB1 in the transfected cells used in (B) were confirmed by Western blot analysis. Mitochondrial enzyme histochemical analysis revealed reduction of succinate dehydrogenase (SDH) and cytochrome oxidase (COX) activity in Purkinje cells of Atxn1-KI mice. The reduced activity of these enzymes was restored in Atxn1-KI;HMGB1 mice. FACS analysis with the well-characterized potentiometric fluorescent dye tetramethylrhodamine methyl ester (TMRM) to quantify the changes of the mitochondrial membrane potential and mitochondrial permeability transition induced by HMGB1. The upper left panel shows experimental procedure, and the upper right panel shows parameters in the following graphs. Non-specific negative control siRNA did not affect TMRM signals, whereas siRNA-A and siRNA-B against HMGB1 substantially reduced a part of transfected HeLa cells. The siRNAs that were used for this analysis were similar to those in , where suppression of HMGB1 by these siRNAs was confirmed. The results from three sets of independent transfection experiments indicated that the mitochondrial membrane potential and mitochondrial permeability transition were changed by the deficiency in HMGB1. Source data are available online for this figure.

Article Snippet: These sections were incubated sequentially with 1% BSA for 20 min, with an anti-HMGB1 antibody (AHM0915, dilution 1:100; ATGen, Seongnam-si, Korea) overnight at 4°C and with mouse anti-IgG antibodies conjugated with 1.4-nm gold particles (#2002, dilution 1:50; Nanoprobes, NY, USA) for 1 h at room temperature.

Techniques: Membrane, Mutagenesis, Transfection, Expressing, Knockdown, Staining, Negative Control, Western Blot, Activity Assay, Permeability

Journal: EMBO Molecular Medicine

Article Title: HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

doi: 10.15252/emmm.201404392

Figure Lengend Snippet: The mitochondrial DNA amplification assay with cerebellar tissue revealed that mitochondrial DNA damage was enhanced in mutant Atxn1-KI mice but reversed in the double-transgenic mice. The number of long cDNA fragments decreases when multiple DNA breaks occur between the primers, while short fragments are more easily amplified. The ratio of the long fragment to the short fragment was decreased in mutant Atxn1-KI mice (right panel). The data are presented as mean ± SD. Statistical analysis involved Student's t -test. The chloramphenicol (CAP) resistance assay of HeLa cells transfected with a control vector (pDsRed), Atxn1-86Q expression vector (pDsRed-Atxn1-86Q) or Atxn1-86Q expression vector + HMGB1 expression vector (pCI-HMGB1). The results showed an increase in mitochondrial DNA damage in mutant Atxn1-expressing HeLa cells. The increase was abrogated by co-expression of HMGB1. The data are presented as mean ± SD. Statistical analysis involved Student's t -test; * P < 0.05. Expression levels of Atxn1 and HMGB1 in transiently transfected HeLa cells used for the CAP assay in (B). Atxn1-86Q was detected with an anti-1C2 antibody. Analysis of mitochondrial genomic DNA using a next-generation sequencer. The cumulative percentages of read counts are plotted against the read length (left panel). The read lengths in the mitochondrial genome are also presented with box plots (right panel). Both analyses indicate that the read length was disproportionally shifted to the shorter fraction in Atxn1-KI mice. The shortening of the read length was reversed in Atxn1-KI;HMGB1 mice. The shift of distribution was analysed statistically using Friedman's test ( P = 2.19 × 10 −31 ) followed by Wilcoxon rank-sum test. P -values were corrected using the Bonferroni method. The P -value was 3.24 × 10 −7 in the comparison between Atxn1-KI mice and background mice (wild-type) and 1.84 × 10 −3 between Atxn1-KI;HMGB1 mice and Atxn1-KI mice. Frequency of mutation in the mitochondrial genome of the three genotypes of mice. The changes of mutation frequency were assessed statistically by Fisher's exact test and the post hoc Bonferroni correction. Mutation frequency was increased in Atxn1-KI mice compared to background mice (wild-type; P = 3.24 × 10 −7 ) but normalized in Atxn1-KI;HMGB1 mice compared to Atxn1-KI mice ( P = 1.84 × 10 −3 ). Source data are available online for this figure.

Article Snippet: These sections were incubated sequentially with 1% BSA for 20 min, with an anti-HMGB1 antibody (AHM0915, dilution 1:100; ATGen, Seongnam-si, Korea) overnight at 4°C and with mouse anti-IgG antibodies conjugated with 1.4-nm gold particles (#2002, dilution 1:50; Nanoprobes, NY, USA) for 1 h at room temperature.

Techniques: DNA Amplification, Mutagenesis, Transgenic Assay, Amplification, Transfection, Control, Plasmid Preparation, Expressing, Comparison

Journal: EMBO Molecular Medicine

Article Title: HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

doi: 10.15252/emmm.201404392

Figure Lengend Snippet: The mitochondrial DNA amplification assay with L929 cells at two time points (10 and 180 min) after X-ray irradiation to induce DNA damage. Amplification of the long and short fragments from the mitochondrial genome was performed at each time point after irradiation in mock-transfected, Atxn1-86Q-expressing and Atxn1-86Q-HMGB1-coexpressing L929 cells that had been transfected 48 h before irradiation. The control cells were transfected with the same amount of empty plasmids. Quantitative analysis of DNA damage at each time point using the ratio between short and long PCR fragments (long/short). Reduction of the ratio indicates enhanced DNA damage. At any time point, DNA damage was induced by mutant Atxn1 and attenuated by HMGB1. The data are presented as mean ± SD. # P < 0.05 in one-way ANOVA followed by post hoc Tukey's HSD test. Reversal of DNA damage between minutes 10 and 180 was evaluated by subtraction of the values. Recovery was very small in Atxn1-expressing cells, but it was normalized by co-expression of HMGB1. The data are presented as mean ± SD. # P < 0.05 in one-way ANOVA followed by post hoc Tukey's HSD test. The extent of DNA damage evaluated using 8-OHdG was equivalent among the three types of transfection. Increased levels of mutant Atxn1 and HMGB1 after transfection were tested in whole-cell extracts and in the mitochondrial fraction. Mutant Atxn1 was equally expressed after two types of transfection, and HMGB1 was upregulated in cells and mitochondria. To verify the results from the mitochondrial fraction, blots with anti-HP1α and α-tubulin antibodies were performed with a positive control. Source data are available online for this figure.

Article Snippet: These sections were incubated sequentially with 1% BSA for 20 min, with an anti-HMGB1 antibody (AHM0915, dilution 1:100; ATGen, Seongnam-si, Korea) overnight at 4°C and with mouse anti-IgG antibodies conjugated with 1.4-nm gold particles (#2002, dilution 1:50; Nanoprobes, NY, USA) for 1 h at room temperature.

Techniques: DNA Amplification, Irradiation, Amplification, Transfection, Expressing, Control, Mutagenesis, Positive Control

Journal: EMBO Molecular Medicine

Article Title: HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

doi: 10.15252/emmm.201404392

Figure Lengend Snippet: Binding of HMGB1 to mitochondrial DNA was evaluated by the chromatin immunoprecipitation (ChIP) assay. The mitochondrial fraction was prepared from HeLa cells expressing pDsRed, pDsRed-Atxn1-33Q or pDsRed-Atxn1-86Q using the Mitochondria Isolation Kit (Thermo Fisher Scientific, IL, USA). Before or after immunoprecipitation with anti-HMGB1 antibody, a mitochondrial DNA fragment (1,849 bp) that bound to HMGB1 was amplified by PCR with the mitochondrial DNA-specific primers. The expected size of the PCR product (1,849 bp) was amplified almost equally from three samples before immunoprecipitation (Input, upper panel). In the samples subjected to immunoprecipitation with an anti-HMGB1 antibody (Output, middle panel), the PCR product was upregulated after the irradiation, indicating that a larger amount of HMGB1 protein binds to mitochondrial DNA after irradiation. The enhanced interaction between HMGB1 and the mitochondrial genome after irradiation was inhibited by Atxn1, especially by Atxn1-86Q. Normal IgG did not co-precipitate mitochondrial DNA (Output, lower panel). Expression levels of DsRed, Atxn1-33Q-DsRed and Atxn1-86Q-DsRed from the experiment in (A) were analysed by Western blotting. Quantitative analysis of mitochondrial DNA damage repair after irradiation. In the mitochondrial fraction prepared from HeLa cells after DNA damage by X-rays (8 Gy), we quantified incorporation of radioisotope-labelled nucleotides into damaged mitochondrial DNA during DNA damage repair. DNA repair in mitochondria was suppressed by addition of an anti-HMGB1 antibody but restored by recombinant HMGB1. Source data are available online for this figure.

Article Snippet: These sections were incubated sequentially with 1% BSA for 20 min, with an anti-HMGB1 antibody (AHM0915, dilution 1:100; ATGen, Seongnam-si, Korea) overnight at 4°C and with mouse anti-IgG antibodies conjugated with 1.4-nm gold particles (#2002, dilution 1:50; Nanoprobes, NY, USA) for 1 h at room temperature.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Expressing, Isolation, Immunoprecipitation, Amplification, Irradiation, Western Blot, Recombinant

Journal: EMBO Molecular Medicine

Article Title: HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

doi: 10.15252/emmm.201404392

Figure Lengend Snippet: Mitochondrial DNA damage repair was evaluated in vivo in mice subjected to X-ray irradiation (20 Gy) under anaesthesia. Cerebellar tissues were collected 10, 180 and 300 min after irradiation. The mitochondrial DNA amplification assay in the cerebellar tissue obtained from wild-type (WT), Atxn1-KI (KI) and Atxn1-KI;HMGB1-Tg double-transgenic (KI+H) mice. Amplification of the long and short fragments from the mitochondrial genome was performed at each time point after irradiation. Quantitative analysis of mitochondrial DNA damage at each time point using the ratio between short and long PCR fragments (long/short). Reduction of the ratio indicates enhanced DNA damage. The data are presented as mean ± SD. # P < 0.05 in one-way ANOVA followed by post hoc Tukey's test. Reversal of mitochondrial DNA damage from 10 min to 180 min or 300 min was evaluated by subtraction of the values. The data are presented as mean ± SD. # P < 0.05 in one-way ANOVA followed by post hoc Tukey's test. The nuclear DNA amplification assay in the cerebellar tissue obtained from wild-type (WT), Atxn1-KI (KI) and Atxn1-KI;HMGB1-Tg double-transgenic (KI+H) mice. Amplification of the long and short fragments from the nuclear genome was performed at each time point after the irradiation; # P < 0.05 in one-way ANOVA followed by post hoc Tukey's test. Quantitative analysis of nuclear DNA damage at each time point using the ratio between short and long PCR fragments (long/short). A reduction in the ratio indicates enhanced DNA damage. The data are presented as mean ± SD. # P < 0.05 in one-way ANOVA followed by post hoc Tukey's test. Reversal of nuclear DNA damage from 10 to 180 min or 300 min was evaluated by subtraction of the values. The data are presented as mean ± SD. # P < 0.05 in one-way ANOVA followed by post hoc Tukey's test. Southern blot analysis of mitochondrial genomic DNA prepared from wild-type (WT), Atxn1-KI or Atxn1-KI;HMGB1-Tg double-transgenic mice. Mitochondrial DNA copy numbers were largely similar among the genotypes, although quality of the mitochondrial DNA was remarkably different. Signal intensity of a 16-kb band corresponding to the intact mitochondrial genome and those of a DNA smear (< 16 kb) corresponding to a degraded mitochondrial genome were quantified using ImageQuant LAS500 (GE Healthcare Life Sciences, Little Chalfont, UK) with ImageQuant TL software. Source data are available online for this figure.

Article Snippet: These sections were incubated sequentially with 1% BSA for 20 min, with an anti-HMGB1 antibody (AHM0915, dilution 1:100; ATGen, Seongnam-si, Korea) overnight at 4°C and with mouse anti-IgG antibodies conjugated with 1.4-nm gold particles (#2002, dilution 1:50; Nanoprobes, NY, USA) for 1 h at room temperature.

Techniques: In Vivo, Irradiation, DNA Amplification, Transgenic Assay, Amplification, Southern Blot, Software

Journal: EMBO Molecular Medicine

Article Title: HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

doi: 10.15252/emmm.201404392

Figure Lengend Snippet: The experimental protocol of the AAV injection. Injection of AAV-GFP to the cerebellar surface (at 5 weeks) efficiently transfected cerebellar tissues with AAV. Fluorescence was detected directly by illumination under a fluorescence stereoscopic microscope at 4 weeks after the injection. The AAV vector for the expression of untagged HMGB1 (AAV-HMGB1) was similarly injected at 5 weeks ( n = 12). GFP expression was not detected because HMGB1 was not tagged. The rotarod performance at 9 weeks (4 weeks after injection) and at 13 weeks (8 weeks after injection) revealed improvement of motor function by AAV-HMGB1 in Atxn1-KI mice after the onset of symptoms. The data from days 1 to 3 are presented as mean ± SD. Statistical analysis involved Student's t -test. Survival curves of Atxn1-KI mice (blue line), AAV-HMGB1-injected Atxn1-KI mice (red line), AAV-GFP-injected Atxn1-KI mice (grey line) and wild-type animals (green line). Relative expression levels of HMGB1 mRNA at 9 weeks were evaluated by quantitative PCR with mRNA prepared from cerebellar tissues of Atxn1-KI mice or AAV-HMGB1-injected Atxn1-KI mice (injected at 5 weeks); * P < 0.05, ** P < 0.01. Expression of HMGB1-GFP and GFP, which had been delivered by intrathecal injection of AAV at 5 weeks, was detected in Purkinje cells at 9 and 32 weeks. The GFP signals were directly acquired using fluorescence microscopy without immunostaining (upper panels). Meanwhile, Purkinje cell numbers were evaluated by immunostaining with an anti-calbindin antibody (lower panels). The decrease in the Purkinje cell number in Atxn1-KI mice was reversed by AAV-HMGB1-GFP even at 32 weeks. Dendrites of Purkinje cells in the molecular cell layer were also normalized morphologically at 9 and 32 weeks. The graphs show quantitative analyses of Purkinje cells of AAV-HMGB1-GFP- or AAV-GFP-injected Atxn1-KI mice at 9 and 32 weeks (mean ± SD, * P < 0.05 both in Student's t -test; # P < 0.05 in one-way ANOVA followed by post hoc Tukey's HSD test). Immunohistochemical analysis of the cerebellum of Atxn1-KI mice injected by AAV-HMGB1-GFP revealed that HMGB1 reduced nuclear DNA damage in Purkinje cells. GFP signals were detected directly without immunostaining. The γH2AX signals were visualized with an anti-γH2AX antibody. The γH2AX signals were reduced in the Purkinje cells of Atxn1-KI mice injected with AAV-HMGB1-GFP compared to the signals in Purkinje cells of AAV-GFP-injected Atxn1-KI mice. Western blotting of the cerebellar tissues also showed that γH2AX was decreased in mutant Atxn1-KI mice injected with AAV-HMGB1-GFP. Quantitative analysis of the bands (mean ± SD) confirmed statistical significance of the difference (Student's t -test, P < 0.05). Infection of AAV-HMGB1-GFP restored the thickness of the molecular layer in mutant Atxn1-KI mice at 9 and 32 weeks. The mean thickness was quantified from 12 visual fields, and the means ± SD were calculated for four mice in the AAV-GFP-injected (GFP) or AAV-HMGB1-GFP-injected (HMGB1) group; * P < 0.05 in Student's t -test. Infection with AAV-HMGB1-GFP reduced mitochondrial DNA damage. The increased proportion of the long PCR fragment amplified from mitochondrial DNA revealed AAV-HMGB1-GFP-induced amelioration of mitochondrial DNA damage. The ratio of the short and long fragments was calculated from their band intensity after amplification in 21 PCR cycles. The data are presented as mean ± SD; * P < 0.05 in Student's t -test ( n = 4). Source data are available online for this figure.

Article Snippet: These sections were incubated sequentially with 1% BSA for 20 min, with an anti-HMGB1 antibody (AHM0915, dilution 1:100; ATGen, Seongnam-si, Korea) overnight at 4°C and with mouse anti-IgG antibodies conjugated with 1.4-nm gold particles (#2002, dilution 1:50; Nanoprobes, NY, USA) for 1 h at room temperature.

Techniques: Injection, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Immunohistochemical staining, Western Blot, Mutagenesis, Infection, Amplification