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  • 99
    Millipore mitochondrial dna
    STING and TLR9 pathways are essential for the cationic liposome-induced activation of pulmonary inflammation. WT , Sting -/- and Tlr9 -/- mice were treated with <t>DOTAP</t> liposomes for 48 h. (A) Representative H E staining of lung sections of mice treated with DOTAP liposomes (n=5); scale bar=50 μm. (B) Inflammatory monocyte (CD45 + CD11b + Ly6C + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (C) Inflammatory neutrophil (CD45 + CD11b + Ly6G + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (D) TNF-α expression in the inflammatory neutrophils (CD45 + CD11b + Ly6G + ) in the lungs of WT and Sting -/- mice (n=3). (E) IL-10 production in inflammatory monocytes (CD45 + CD11b + Ly6C + ) in the lungs of WT and Sting -/- mice (n=3). (F) Neutrophils were isolated from the bone marrow of WT and Sting -/- mice and were stimulated with necrotic lung cells or mtDNA (G) for 4 h in the presence of brefeldin A. TNF-α expression in inflammatory neutrophils (CD45 + CD11b + Ly6G + ) was determined by flow cytometry, (n=3). (H) Monocytes were isolated from the bone marrow of WT, Tlr9 -/- and Sting -/- mice and were cultured with mitochondrial <t>DNA</t> (5 μg/mL) at a concentration of 5×10 6 cells/mL; EIA assays were conducted for PGE 2 expression in supernatants (n=3). (I) Western blot analysis was performed. (J) The freshly isolated monocytes were cultured with mtDNA (5 μg/mL) at a concentration of 5×10 6 cells/mL. The inhibitors PD98059 (30 μM), SB203580 (10 μM), BAY117082 (10 μM) and indomethacin (10 μM) were added to the cells, and the cells were incubated for 2 h at 37 °C, then, Western blot analysis was performed. Data are representative of three independent experiments, and the results are expressed as the mean ± S.E.M. Statistical comparisons were performed using Student's t-test or Dunnet's t-test ( *P
    Mitochondrial Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    BioVision mitochondrial dna mtdna
    Selective amino acid restriction induced mitochondrial <t>DNA</t> damage in DU145 cells and decreased damage in PC3 cells. The cells were treated with specific amino acid-deprived medium for 4 days and the mitochondrial DNA <t>(mtDNA)</t> was determined on days 2 to
    Mitochondrial Dna Mtdna, supplied by BioVision, used in various techniques. Bioz Stars score: 95/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam mitochondrial dna mtdna
    Inosine incorporation into nucleic acids in human and mouse cells lacking functional ITPase. (A) Bar chart showing a significantly increased inosine base content of RNA in lymphoblastoid cell lines (LCLs) derived from an affected individual (5196 III:3) as compared to that derived from her mother (5196 II:2) (B) Bar chart showing significantly increased inosine base content of RNA in Itpa -null mouse embryonic stem (ES) cells as compared to control ES cells. (C) Bar chart showing increased inosine base content of RNA derived from Itpa -null tissue as compared to controls. Inosine content is significantly higher in RNA derived from Itpa -null hearts than that stage-matched control hearts. There was no significant (ns) difference in IMP content in RNA derived from Itpa -null compared to control kidneys. Error bars ±SEM. (D) Alkaline-gel electrophoresis of total <t>DNA</t> and <t>mtDNA</t> extracted from mouse ES cells untreated or treated with bacterial endonuclease V (EndoV). All lanes shown are on the same gel, and these data are representative of three independent experiments. (E) Densitometry of gels shown in D does not identify any difference between control (green lines) and Itpa -null (red lines) cells for genomic DNA (top panel) but for mtDNA (bottom panel) there is a shift in the migration pattern in the Itpa -null cells suggestive of an increase EndoV digestion compared to the controls. (F) Long-range PCR (LR-PCR) of the mitochondrial genome shows no evidence for increased deletions in Itpa -null ES cells as compared to controls. The data shown are representative of three independent experiments. The primers used are listed in S2 Table . (G) Quantitative RT-PCR (qPCR) on total DNA shows that ratios of mtDNA to genomic DNA are comparable between control and Itpa -null cells. The data shown are derived from analysis of six individual DNA preparations per genotype, each analysed in triplicate. All the primers used are listed in S2 Table . (H,I) Alkaline comet assays on LCLs derived from an affected individual (5196 III:3) and her mother (5196 II:2) and null and parental mouse ESC respectively with cells exposed to hydrogen peroxide as a positive control. Neither cell type shows evidence for increase single or double strand breaks in genomic DNA. Quantitation of DNA damage is by Olive tail moment (the product of the tail length and the fraction of total DNA in the tail) and is a measure of both the extent of DNA fragmentation and size of fragmented DNA.
    Mitochondrial Dna Mtdna, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa human mitochondrial dna mtdna
    N-Myc overexpression increased mitochondrial biogenesis. ( a ) Whole cell lysates (WCL) from SH and SH-N-Myc cells were collected and used for western analysis with N-Myc antibodies that showed N-Myc was highly overexpressed in our model. ( b ) WCL were used to measure expression of the global mitochondrial regulators PGC1-a and TFAM. Both are upregulated in SH-N-Myc. ( c ) Cells at mid-logarithmic phase were stained with MitoTracker Green and measured by flow cytometry. A representative curve is shown. ( d ) A qPCR-based assay was used to measure mitochondrial <t>DNA</t> copy number using genomic DNA content as the control. Four separate experiments were performed with each cell line being measured at least in triplicate each time. Error bars show standard error of the experiments. P values: * P
    Human Mitochondrial Dna Mtdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad mitochondrial dna mitochondrial dna
    N-Myc overexpression increased mitochondrial biogenesis. ( a ) Whole cell lysates (WCL) from SH and SH-N-Myc cells were collected and used for western analysis with N-Myc antibodies that showed N-Myc was highly overexpressed in our model. ( b ) WCL were used to measure expression of the global mitochondrial regulators PGC1-a and TFAM. Both are upregulated in SH-N-Myc. ( c ) Cells at mid-logarithmic phase were stained with MitoTracker Green and measured by flow cytometry. A representative curve is shown. ( d ) A qPCR-based assay was used to measure mitochondrial <t>DNA</t> copy number using genomic DNA content as the control. Four separate experiments were performed with each cell line being measured at least in triplicate each time. Error bars show standard error of the experiments. P values: * P
    Mitochondrial Dna Mitochondrial Dna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa mitochondrial dna mtdna
    Spatial sampling of a Barrett's segment and associated oesophageal adenocarcinoma (OAC). (A) Overview of the opened resection specimen shows columnar metaplasia across the gastro-oesophageal junction (arrows) and a nodular OAC (arrowhead). The rectangular box indicates the longitudinal strip that was sampled. (B) H E-stained cryostat section (left) of the longitudinal strip across the gastro-oesophageal junction reveals columnar metaplasia of the distal oesophagus (arrows) and an OAC at the squamocolumnar junction (arrowhead). Submucosal gland complex (asterisk) confirms the oesophageal origin. Cytochrome c oxidase (CCO) staining of this longitudinal strip (right) shows several discontinuous epithelial patches that are CCO-deficient. The OAC is also CCO-deficient. (B) and (C) images are taken at same magnification (see scalebar). The boxed area is shown in detail in (C). (C) Overview and high-power photomicrographs of one CCO-deficient epithelial patch and the associated OAC. Note the mosaic spread of the CCO-deficient clone within the background mucosa. There are no architectural or cytological features of dysplasia. OAC shows atypical, cribriform glands, which penetrate the pre-existent muscularis mucosae. (D) Deep next-generation mitochondrial <t>DNA</t> <t>(mtDNA)</t> sequencing reveals unique mtDNA mutations within the CCO-deficient epithelial patch shown in (C). Barchart shows the variant allele fractions (VAFs) of the mutations in control (stroma) sample and in material from the clonal expansion, mtDNA mutations are indicated. The 16290C > T mutation was selected for further analysis. (E) Sanger resequencing shows that spatially distinct CCO-deficient epithelial patches and the OAC carry the same 16290C > T mtDNA mutation (see also table 1 ). CCO-proficient epithelium does not carry this genetic lineage marker. (F) Consecutive sections of neighbouring glands showing cardia-type metaplasia and intestinal metaplasia. Top left: H E staining shows absence of goblet cell differentiation in non-dysplastic cardia-type epithelium (marked by arrowhead), whereas the neighbouring intestinal metaplasia shows abundant goblet cells (marked by asterisk). Top right: clonal loss of CCO activity in cardia-type epithelium (marked by arrowhead). Bottom left: CDX2 staining confirms absence of intestinalisation in CCO-deficient cardia-type metaplasia. Strong nuclear labelling is seen in neighbouring intestinal metaplasia (marked by arrowhead). Bottom right: low proliferative activity as shown by Ki67 proliferation marker stain, consistent with morphological absence of dysplasia. Arrowhead points to positive nuclear labelling.
    Mitochondrial Dna Mtdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore mitochondrial dna mtdna
    Spatial sampling of a Barrett's segment and associated oesophageal adenocarcinoma (OAC). (A) Overview of the opened resection specimen shows columnar metaplasia across the gastro-oesophageal junction (arrows) and a nodular OAC (arrowhead). The rectangular box indicates the longitudinal strip that was sampled. (B) H E-stained cryostat section (left) of the longitudinal strip across the gastro-oesophageal junction reveals columnar metaplasia of the distal oesophagus (arrows) and an OAC at the squamocolumnar junction (arrowhead). Submucosal gland complex (asterisk) confirms the oesophageal origin. Cytochrome c oxidase (CCO) staining of this longitudinal strip (right) shows several discontinuous epithelial patches that are CCO-deficient. The OAC is also CCO-deficient. (B) and (C) images are taken at same magnification (see scalebar). The boxed area is shown in detail in (C). (C) Overview and high-power photomicrographs of one CCO-deficient epithelial patch and the associated OAC. Note the mosaic spread of the CCO-deficient clone within the background mucosa. There are no architectural or cytological features of dysplasia. OAC shows atypical, cribriform glands, which penetrate the pre-existent muscularis mucosae. (D) Deep next-generation mitochondrial <t>DNA</t> <t>(mtDNA)</t> sequencing reveals unique mtDNA mutations within the CCO-deficient epithelial patch shown in (C). Barchart shows the variant allele fractions (VAFs) of the mutations in control (stroma) sample and in material from the clonal expansion, mtDNA mutations are indicated. The 16290C > T mutation was selected for further analysis. (E) Sanger resequencing shows that spatially distinct CCO-deficient epithelial patches and the OAC carry the same 16290C > T mtDNA mutation (see also table 1 ). CCO-proficient epithelium does not carry this genetic lineage marker. (F) Consecutive sections of neighbouring glands showing cardia-type metaplasia and intestinal metaplasia. Top left: H E staining shows absence of goblet cell differentiation in non-dysplastic cardia-type epithelium (marked by arrowhead), whereas the neighbouring intestinal metaplasia shows abundant goblet cells (marked by asterisk). Top right: clonal loss of CCO activity in cardia-type epithelium (marked by arrowhead). Bottom left: CDX2 staining confirms absence of intestinalisation in CCO-deficient cardia-type metaplasia. Strong nuclear labelling is seen in neighbouring intestinal metaplasia (marked by arrowhead). Bottom right: low proliferative activity as shown by Ki67 proliferation marker stain, consistent with morphological absence of dysplasia. Arrowhead points to positive nuclear labelling.
    Mitochondrial Dna Mtdna, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Red Sea mitochondrial dna mtdna
    Spatial sampling of a Barrett's segment and associated oesophageal adenocarcinoma (OAC). (A) Overview of the opened resection specimen shows columnar metaplasia across the gastro-oesophageal junction (arrows) and a nodular OAC (arrowhead). The rectangular box indicates the longitudinal strip that was sampled. (B) H E-stained cryostat section (left) of the longitudinal strip across the gastro-oesophageal junction reveals columnar metaplasia of the distal oesophagus (arrows) and an OAC at the squamocolumnar junction (arrowhead). Submucosal gland complex (asterisk) confirms the oesophageal origin. Cytochrome c oxidase (CCO) staining of this longitudinal strip (right) shows several discontinuous epithelial patches that are CCO-deficient. The OAC is also CCO-deficient. (B) and (C) images are taken at same magnification (see scalebar). The boxed area is shown in detail in (C). (C) Overview and high-power photomicrographs of one CCO-deficient epithelial patch and the associated OAC. Note the mosaic spread of the CCO-deficient clone within the background mucosa. There are no architectural or cytological features of dysplasia. OAC shows atypical, cribriform glands, which penetrate the pre-existent muscularis mucosae. (D) Deep next-generation mitochondrial <t>DNA</t> <t>(mtDNA)</t> sequencing reveals unique mtDNA mutations within the CCO-deficient epithelial patch shown in (C). Barchart shows the variant allele fractions (VAFs) of the mutations in control (stroma) sample and in material from the clonal expansion, mtDNA mutations are indicated. The 16290C > T mutation was selected for further analysis. (E) Sanger resequencing shows that spatially distinct CCO-deficient epithelial patches and the OAC carry the same 16290C > T mtDNA mutation (see also table 1 ). CCO-proficient epithelium does not carry this genetic lineage marker. (F) Consecutive sections of neighbouring glands showing cardia-type metaplasia and intestinal metaplasia. Top left: H E staining shows absence of goblet cell differentiation in non-dysplastic cardia-type epithelium (marked by arrowhead), whereas the neighbouring intestinal metaplasia shows abundant goblet cells (marked by asterisk). Top right: clonal loss of CCO activity in cardia-type epithelium (marked by arrowhead). Bottom left: CDX2 staining confirms absence of intestinalisation in CCO-deficient cardia-type metaplasia. Strong nuclear labelling is seen in neighbouring intestinal metaplasia (marked by arrowhead). Bottom right: low proliferative activity as shown by Ki67 proliferation marker stain, consistent with morphological absence of dysplasia. Arrowhead points to positive nuclear labelling.
    Mitochondrial Dna Mtdna, supplied by Red Sea, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Qiagen repli g mitochondrial dna kit
    Quantification of mtDNA enrichment by qPCR. A. Quantification after nuclear <t>DNA</t> removal using exonuclease V (ExoV) as indicated. B. Quantification after mtDNA amplification using MDA. The results are given for DNA untreated by exoV (No ExoV), or after two conditions of ExoV digestion as indicated. <t>REPLI-g</t> was performed using primers at a concentration of 0.2 µM or 1.0 µM.
    Repli G Mitochondrial Dna Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 88/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Biotechnology Information mitochondrial dna mtdna sequences
    Analysis of direct repeat (DR) motifs in mitochondrial genomes. Abundance, distribution and free energy of DRs in <t>mtDNA</t> and random <t>DNA</t> sequences ( n = 100) with the same base composition (R1) as corresponding mtDNA. (A) Frequency of DR pairs (≥6 bp) in the mtDNA of human, rhesus monkey, mouse and rat. The DR frequency is normalized with respect to the mtDNA length in each species. (B) The distribution of left and right DR sequences in the minor and major arcs of human mtDNA and the mean distribution of DRs in R1 random sequences. (C) DR sizes and DR free energies in human mtDNA and the corresponding R1 random sequences. The lower the free energy of a DR, i.e. the more negative, the more stable is the DNA duplex formed. (D) Distribution of free energies of DNA duplex formed by DRs (≥6 bp) in native mtDNA and random sequences (R1) of human, rhesus monkey, mouse and rat.
    Mitochondrial Dna Mtdna Sequences, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher mitochondrial dna mtdna mutations
    Exclusion of mitochondrial <t>DNA</t> <t>(mtDNA)</t> mutations occurring in the germline or in early embryogenesis. ( A ) Somatic mtDNA frequency (mtDNA mutations present in colon only) measured by NGS. There was a significantly higher mutation frequency in the subjects > 70 years (P = 0.0351, unpaired t-test). ( B ) Percentage of synonymous/polymorphic variants and non-synonymous mtDNA mutations which were of germline or early embryological origin compared to those which were somatic in adults
    Mitochondrial Dna Mtdna Mutations, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bioedit Company mitochondrial dna mtdna sequences
    Exclusion of mitochondrial <t>DNA</t> <t>(mtDNA)</t> mutations occurring in the germline or in early embryogenesis. ( A ) Somatic mtDNA frequency (mtDNA mutations present in colon only) measured by NGS. There was a significantly higher mutation frequency in the subjects > 70 years (P = 0.0351, unpaired t-test). ( B ) Percentage of synonymous/polymorphic variants and non-synonymous mtDNA mutations which were of germline or early embryological origin compared to those which were somatic in adults
    Mitochondrial Dna Mtdna Sequences, supplied by Bioedit Company, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc mtdna
    Mitochondrial BER deficient mice do not accumulate point mutations to <t>mtDNA</t> after five generations of consecutive breeding. ( A ) Breeding scheme to accumulate mutations into mtDNA and study germ line mutations. Homozygous Mutyh dMTS × Ogg1 dMTS female mice were bred with homo- or heterozygous Mutyh dMTS × Ogg1 dMTS male mice for five consecutive generations. To minimize the nuclear effects, heterozygote male mice were also used in the breedings. N1–N5 indicates the generations of breeding. ( B ) Mutation load of mtDNA with <t>Illumina</t> sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. The sequencing was carried out from purified mtDNA from liver. Data is quality filtered and minimum variant allele frequency is set to 0.5%. In unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. In total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (++ n = 6, pp n = 2, 10–13 week old) and gray circles indicate samples from homozygous Mutyh dMTS × Ogg1 dMTS mice (dd dd, n = 8, 10–15 week old). Horizontal lines represent means. C. Mutation profile of mtDNA with Illumina sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. Samples as in B. Horizontal lines represent means. For only quality filtered data see Supplementary Figure S3 .
    Mtdna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Quest Diagnostics mtdna
    Mitochondrial BER deficient mice do not accumulate point mutations to <t>mtDNA</t> after five generations of consecutive breeding. ( A ) Breeding scheme to accumulate mutations into mtDNA and study germ line mutations. Homozygous Mutyh dMTS × Ogg1 dMTS female mice were bred with homo- or heterozygous Mutyh dMTS × Ogg1 dMTS male mice for five consecutive generations. To minimize the nuclear effects, heterozygote male mice were also used in the breedings. N1–N5 indicates the generations of breeding. ( B ) Mutation load of mtDNA with <t>Illumina</t> sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. The sequencing was carried out from purified mtDNA from liver. Data is quality filtered and minimum variant allele frequency is set to 0.5%. In unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. In total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (++ n = 6, pp n = 2, 10–13 week old) and gray circles indicate samples from homozygous Mutyh dMTS × Ogg1 dMTS mice (dd dd, n = 8, 10–15 week old). Horizontal lines represent means. C. Mutation profile of mtDNA with Illumina sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. Samples as in B. Horizontal lines represent means. For only quality filtered data see Supplementary Figure S3 .
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    STING and TLR9 pathways are essential for the cationic liposome-induced activation of pulmonary inflammation. WT , Sting -/- and Tlr9 -/- mice were treated with DOTAP liposomes for 48 h. (A) Representative H E staining of lung sections of mice treated with DOTAP liposomes (n=5); scale bar=50 μm. (B) Inflammatory monocyte (CD45 + CD11b + Ly6C + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (C) Inflammatory neutrophil (CD45 + CD11b + Ly6G + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (D) TNF-α expression in the inflammatory neutrophils (CD45 + CD11b + Ly6G + ) in the lungs of WT and Sting -/- mice (n=3). (E) IL-10 production in inflammatory monocytes (CD45 + CD11b + Ly6C + ) in the lungs of WT and Sting -/- mice (n=3). (F) Neutrophils were isolated from the bone marrow of WT and Sting -/- mice and were stimulated with necrotic lung cells or mtDNA (G) for 4 h in the presence of brefeldin A. TNF-α expression in inflammatory neutrophils (CD45 + CD11b + Ly6G + ) was determined by flow cytometry, (n=3). (H) Monocytes were isolated from the bone marrow of WT, Tlr9 -/- and Sting -/- mice and were cultured with mitochondrial DNA (5 μg/mL) at a concentration of 5×10 6 cells/mL; EIA assays were conducted for PGE 2 expression in supernatants (n=3). (I) Western blot analysis was performed. (J) The freshly isolated monocytes were cultured with mtDNA (5 μg/mL) at a concentration of 5×10 6 cells/mL. The inhibitors PD98059 (30 μM), SB203580 (10 μM), BAY117082 (10 μM) and indomethacin (10 μM) were added to the cells, and the cells were incubated for 2 h at 37 °C, then, Western blot analysis was performed. Data are representative of three independent experiments, and the results are expressed as the mean ± S.E.M. Statistical comparisons were performed using Student's t-test or Dunnet's t-test ( *P

    Journal: Theranostics

    Article Title: Negative regulation of cationic nanoparticle-induced inflammatory toxicity through the increased production of prostaglandin E2 via mitochondrial DNA-activated Ly6C+ monocytes

    doi: 10.7150/thno.21693

    Figure Lengend Snippet: STING and TLR9 pathways are essential for the cationic liposome-induced activation of pulmonary inflammation. WT , Sting -/- and Tlr9 -/- mice were treated with DOTAP liposomes for 48 h. (A) Representative H E staining of lung sections of mice treated with DOTAP liposomes (n=5); scale bar=50 μm. (B) Inflammatory monocyte (CD45 + CD11b + Ly6C + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (C) Inflammatory neutrophil (CD45 + CD11b + Ly6G + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (D) TNF-α expression in the inflammatory neutrophils (CD45 + CD11b + Ly6G + ) in the lungs of WT and Sting -/- mice (n=3). (E) IL-10 production in inflammatory monocytes (CD45 + CD11b + Ly6C + ) in the lungs of WT and Sting -/- mice (n=3). (F) Neutrophils were isolated from the bone marrow of WT and Sting -/- mice and were stimulated with necrotic lung cells or mtDNA (G) for 4 h in the presence of brefeldin A. TNF-α expression in inflammatory neutrophils (CD45 + CD11b + Ly6G + ) was determined by flow cytometry, (n=3). (H) Monocytes were isolated from the bone marrow of WT, Tlr9 -/- and Sting -/- mice and were cultured with mitochondrial DNA (5 μg/mL) at a concentration of 5×10 6 cells/mL; EIA assays were conducted for PGE 2 expression in supernatants (n=3). (I) Western blot analysis was performed. (J) The freshly isolated monocytes were cultured with mtDNA (5 μg/mL) at a concentration of 5×10 6 cells/mL. The inhibitors PD98059 (30 μM), SB203580 (10 μM), BAY117082 (10 μM) and indomethacin (10 μM) were added to the cells, and the cells were incubated for 2 h at 37 °C, then, Western blot analysis was performed. Data are representative of three independent experiments, and the results are expressed as the mean ± S.E.M. Statistical comparisons were performed using Student's t-test or Dunnet's t-test ( *P

    Article Snippet: Mice were injected with DOTAP liposomes (25 mg/kg), necrotic lung cells (1×106 cells/mouse) or mitochondrial DNA (5 μg/mouse) and were treated with intravenous injections of diMePGE2 (10 or 100 μg/kg body weight) (Sigma).

    Techniques: Activation Assay, Mouse Assay, Staining, Expressing, Isolation, Flow Cytometry, Cytometry, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

    Selective amino acid restriction induced mitochondrial DNA damage in DU145 cells and decreased damage in PC3 cells. The cells were treated with specific amino acid-deprived medium for 4 days and the mitochondrial DNA (mtDNA) was determined on days 2 to

    Journal: Oncology Letters

    Article Title: Differential effects of specific amino acid restriction on glucose metabolism, reduction/oxidation status and mitochondrial damage in DU145 and PC3 prostate cancer cells

    doi: 10.3892/ol.2011.237

    Figure Lengend Snippet: Selective amino acid restriction induced mitochondrial DNA damage in DU145 cells and decreased damage in PC3 cells. The cells were treated with specific amino acid-deprived medium for 4 days and the mitochondrial DNA (mtDNA) was determined on days 2 to

    Article Snippet: Mitochondrial DNA (mtDNA) was isolated (Mitochondrial DNA isolation kit; Biovision) and quantified (Quant-It PicoGreen dsDNA reagent and kits; Molecular Probes Inc.).

    Techniques:

    Inosine incorporation into nucleic acids in human and mouse cells lacking functional ITPase. (A) Bar chart showing a significantly increased inosine base content of RNA in lymphoblastoid cell lines (LCLs) derived from an affected individual (5196 III:3) as compared to that derived from her mother (5196 II:2) (B) Bar chart showing significantly increased inosine base content of RNA in Itpa -null mouse embryonic stem (ES) cells as compared to control ES cells. (C) Bar chart showing increased inosine base content of RNA derived from Itpa -null tissue as compared to controls. Inosine content is significantly higher in RNA derived from Itpa -null hearts than that stage-matched control hearts. There was no significant (ns) difference in IMP content in RNA derived from Itpa -null compared to control kidneys. Error bars ±SEM. (D) Alkaline-gel electrophoresis of total DNA and mtDNA extracted from mouse ES cells untreated or treated with bacterial endonuclease V (EndoV). All lanes shown are on the same gel, and these data are representative of three independent experiments. (E) Densitometry of gels shown in D does not identify any difference between control (green lines) and Itpa -null (red lines) cells for genomic DNA (top panel) but for mtDNA (bottom panel) there is a shift in the migration pattern in the Itpa -null cells suggestive of an increase EndoV digestion compared to the controls. (F) Long-range PCR (LR-PCR) of the mitochondrial genome shows no evidence for increased deletions in Itpa -null ES cells as compared to controls. The data shown are representative of three independent experiments. The primers used are listed in S2 Table . (G) Quantitative RT-PCR (qPCR) on total DNA shows that ratios of mtDNA to genomic DNA are comparable between control and Itpa -null cells. The data shown are derived from analysis of six individual DNA preparations per genotype, each analysed in triplicate. All the primers used are listed in S2 Table . (H,I) Alkaline comet assays on LCLs derived from an affected individual (5196 III:3) and her mother (5196 II:2) and null and parental mouse ESC respectively with cells exposed to hydrogen peroxide as a positive control. Neither cell type shows evidence for increase single or double strand breaks in genomic DNA. Quantitation of DNA damage is by Olive tail moment (the product of the tail length and the fraction of total DNA in the tail) and is a measure of both the extent of DNA fragmentation and size of fragmented DNA.

    Journal: PLoS Genetics

    Article Title: ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy

    doi: 10.1371/journal.pgen.1007605

    Figure Lengend Snippet: Inosine incorporation into nucleic acids in human and mouse cells lacking functional ITPase. (A) Bar chart showing a significantly increased inosine base content of RNA in lymphoblastoid cell lines (LCLs) derived from an affected individual (5196 III:3) as compared to that derived from her mother (5196 II:2) (B) Bar chart showing significantly increased inosine base content of RNA in Itpa -null mouse embryonic stem (ES) cells as compared to control ES cells. (C) Bar chart showing increased inosine base content of RNA derived from Itpa -null tissue as compared to controls. Inosine content is significantly higher in RNA derived from Itpa -null hearts than that stage-matched control hearts. There was no significant (ns) difference in IMP content in RNA derived from Itpa -null compared to control kidneys. Error bars ±SEM. (D) Alkaline-gel electrophoresis of total DNA and mtDNA extracted from mouse ES cells untreated or treated with bacterial endonuclease V (EndoV). All lanes shown are on the same gel, and these data are representative of three independent experiments. (E) Densitometry of gels shown in D does not identify any difference between control (green lines) and Itpa -null (red lines) cells for genomic DNA (top panel) but for mtDNA (bottom panel) there is a shift in the migration pattern in the Itpa -null cells suggestive of an increase EndoV digestion compared to the controls. (F) Long-range PCR (LR-PCR) of the mitochondrial genome shows no evidence for increased deletions in Itpa -null ES cells as compared to controls. The data shown are representative of three independent experiments. The primers used are listed in S2 Table . (G) Quantitative RT-PCR (qPCR) on total DNA shows that ratios of mtDNA to genomic DNA are comparable between control and Itpa -null cells. The data shown are derived from analysis of six individual DNA preparations per genotype, each analysed in triplicate. All the primers used are listed in S2 Table . (H,I) Alkaline comet assays on LCLs derived from an affected individual (5196 III:3) and her mother (5196 II:2) and null and parental mouse ESC respectively with cells exposed to hydrogen peroxide as a positive control. Neither cell type shows evidence for increase single or double strand breaks in genomic DNA. Quantitation of DNA damage is by Olive tail moment (the product of the tail length and the fraction of total DNA in the tail) and is a measure of both the extent of DNA fragmentation and size of fragmented DNA.

    Article Snippet: Detection of inosine bases in hydrolysed RNA and DNA Cellular RNA and DNA were purified using RNAeasy (Qiagen) and BACC2 (GE Healthcare) kits respectively. mtDNA isolation was performed using a mitochondrial DNA isolation kit (Abcam).

    Techniques: Functional Assay, Derivative Assay, Nucleic Acid Electrophoresis, Migration, Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Positive Control, Quantitation Assay

    Damaged mitochondrial DNA activates ZBP1/TBK1/IRF3 signaling pathway. Interaction between BrDU-labelled mtDNA and TFAM ( A ) and BrDU-labelled mtDNA and ZBP1 ( B ) in control and GOx-treated cells at 1 h. ( C ) Interaction between ZBP1/TBK1, IRF3/TBK1, ZBP1/P-Tyr and ZBP1/P-Ser in control and GOx-treated cells at 1 h. ( D ) Interaction between IRF3/TBK1 in pre-treated with 3 μM of CsA in GOx-treated BEAS 2B cells. ( E ) Level of ZBP1 depletion, shown by Western blotting. ( F ) Expression of IL-6 and IL-8 in unstressed and GOx-treated (0.006 U/ml for 1 h) in control and ZBP1-depleted BEAS2B cells. Representative images of n = 3 independent experiments are shown. Data represent average ± SEM of n = 5 biological replicates. *p

    Journal: Scientific Reports

    Article Title: Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells

    doi: 10.1038/s41598-018-19216-1

    Figure Lengend Snippet: Damaged mitochondrial DNA activates ZBP1/TBK1/IRF3 signaling pathway. Interaction between BrDU-labelled mtDNA and TFAM ( A ) and BrDU-labelled mtDNA and ZBP1 ( B ) in control and GOx-treated cells at 1 h. ( C ) Interaction between ZBP1/TBK1, IRF3/TBK1, ZBP1/P-Tyr and ZBP1/P-Ser in control and GOx-treated cells at 1 h. ( D ) Interaction between IRF3/TBK1 in pre-treated with 3 μM of CsA in GOx-treated BEAS 2B cells. ( E ) Level of ZBP1 depletion, shown by Western blotting. ( F ) Expression of IL-6 and IL-8 in unstressed and GOx-treated (0.006 U/ml for 1 h) in control and ZBP1-depleted BEAS2B cells. Representative images of n = 3 independent experiments are shown. Data represent average ± SEM of n = 5 biological replicates. *p

    Article Snippet: MtDNA was isolated from mouse liver by using Mitochondrial DNA Isolation Kit (abcam, ab65321).

    Techniques: Western Blot, Expressing

    Oxidatively stressed cells induce release of damaged mtDNA via exosomes. ( A ) Comparison of mitochondrial and nuclear DNA in exosomal fraction isolated at 24 h from BEAS 2B cells treated with 0.006 U/ml GOx. ( B ) Time-dependent release of the mtDNA from control and BEAS 2B cells treated with 0.006 U/ml of GOx. ( C ) Protein level of CD63, marker of exosomes, in BEAS 2B cell extract and in exosomal fraction. MtDNA release from cells pretreated for 1 h with ( D ) 300 nM of Mito-TEMPO; ( E ) 3 μM cyclosporine-A or ( F ) 3 μM colchicine in control and 0.006 U/ml of GOx-treated BEAS 2B cells. Data represent average ± SEM of n = 5 biological replicates. Representative images of n = 3 independent experiments are shown. **p

    Journal: Scientific Reports

    Article Title: Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells

    doi: 10.1038/s41598-018-19216-1

    Figure Lengend Snippet: Oxidatively stressed cells induce release of damaged mtDNA via exosomes. ( A ) Comparison of mitochondrial and nuclear DNA in exosomal fraction isolated at 24 h from BEAS 2B cells treated with 0.006 U/ml GOx. ( B ) Time-dependent release of the mtDNA from control and BEAS 2B cells treated with 0.006 U/ml of GOx. ( C ) Protein level of CD63, marker of exosomes, in BEAS 2B cell extract and in exosomal fraction. MtDNA release from cells pretreated for 1 h with ( D ) 300 nM of Mito-TEMPO; ( E ) 3 μM cyclosporine-A or ( F ) 3 μM colchicine in control and 0.006 U/ml of GOx-treated BEAS 2B cells. Data represent average ± SEM of n = 5 biological replicates. Representative images of n = 3 independent experiments are shown. **p

    Article Snippet: MtDNA was isolated from mouse liver by using Mitochondrial DNA Isolation Kit (abcam, ab65321).

    Techniques: Isolation, Marker

    Mitochondrial DNA is released into the bronchoalveolar lavage fluid as an early event in a murine model of cigarette smoke induced lung injury. Early presence of mtDNA ( A ) but not of nuclear DNA ( B ) in BALF of mice exposed to cigarette smoke induced lung injury. MtDNA content is depleted ( C ) and mtDNA integrity is impaired ( D ) in the lung tissue of smoke injured mice. 6–8 animals were used for each experimental end-point. Data represent average ± SEM. **p

    Journal: Scientific Reports

    Article Title: Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells

    doi: 10.1038/s41598-018-19216-1

    Figure Lengend Snippet: Mitochondrial DNA is released into the bronchoalveolar lavage fluid as an early event in a murine model of cigarette smoke induced lung injury. Early presence of mtDNA ( A ) but not of nuclear DNA ( B ) in BALF of mice exposed to cigarette smoke induced lung injury. MtDNA content is depleted ( C ) and mtDNA integrity is impaired ( D ) in the lung tissue of smoke injured mice. 6–8 animals were used for each experimental end-point. Data represent average ± SEM. **p

    Article Snippet: MtDNA was isolated from mouse liver by using Mitochondrial DNA Isolation Kit (abcam, ab65321).

    Techniques: Mouse Assay

    Inflammation respond to mitochondrial DNA in vivo . ( A ) Inflammatory response triggered in BALF of naïve mice at 24 h post intratracheal administration of 1 μg of isolated mtDNA. ( B ) Time-dependent expression of IL-1α in BEAS 2B cells incubated 100 ng of isolated mtDNA. ( C ) Model of inflammation induced by mtDNA/ZBP1 pathway. Data represent average ± SEM using n = 6–8 animals or n = 3 for biological replicates per experimental end-point. *p

    Journal: Scientific Reports

    Article Title: Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells

    doi: 10.1038/s41598-018-19216-1

    Figure Lengend Snippet: Inflammation respond to mitochondrial DNA in vivo . ( A ) Inflammatory response triggered in BALF of naïve mice at 24 h post intratracheal administration of 1 μg of isolated mtDNA. ( B ) Time-dependent expression of IL-1α in BEAS 2B cells incubated 100 ng of isolated mtDNA. ( C ) Model of inflammation induced by mtDNA/ZBP1 pathway. Data represent average ± SEM using n = 6–8 animals or n = 3 for biological replicates per experimental end-point. *p

    Article Snippet: MtDNA was isolated from mouse liver by using Mitochondrial DNA Isolation Kit (abcam, ab65321).

    Techniques: In Vivo, Mouse Assay, Isolation, Expressing, Incubation

    Non-cytotoxic levels of oxidative stress induce mitochondrial DNA depletion, impair cellular bioenergetics and stimulate inflammation. BEAS2B cells were treated with several concentrations of GOx for 1 h and immediately post-challenge as well as 24 h later were measured: ( A ) mitochondrial DNA integrity, ( B ) mitochondrial DNA content, ( C ) apoptotic/necrotic cell death; ( D ) mitochondrial respiration; ( E ) glycolysis. ( F ) 3-D reconstruction of cellular morphology of BEAS2B at 24 h after GOx-treatment was visualized using ATPA-specific antibody (green), β-tubulin (red) and nucleus (DAPI, blue). ( G ) Amount of pro-inflammatory mediators in medium of BEAS2B cells measured at 24 h post GOx-treatment. Data represent average ± SEM of n = 5 biological replicates. ( H ) Lack of enhanced production of IL-6 in BEAS 2B cells stimulated with inactive GOx at 24 h. Representative data of n = 3 independent experiments are shown. *p

    Journal: Scientific Reports

    Article Title: Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells

    doi: 10.1038/s41598-018-19216-1

    Figure Lengend Snippet: Non-cytotoxic levels of oxidative stress induce mitochondrial DNA depletion, impair cellular bioenergetics and stimulate inflammation. BEAS2B cells were treated with several concentrations of GOx for 1 h and immediately post-challenge as well as 24 h later were measured: ( A ) mitochondrial DNA integrity, ( B ) mitochondrial DNA content, ( C ) apoptotic/necrotic cell death; ( D ) mitochondrial respiration; ( E ) glycolysis. ( F ) 3-D reconstruction of cellular morphology of BEAS2B at 24 h after GOx-treatment was visualized using ATPA-specific antibody (green), β-tubulin (red) and nucleus (DAPI, blue). ( G ) Amount of pro-inflammatory mediators in medium of BEAS2B cells measured at 24 h post GOx-treatment. Data represent average ± SEM of n = 5 biological replicates. ( H ) Lack of enhanced production of IL-6 in BEAS 2B cells stimulated with inactive GOx at 24 h. Representative data of n = 3 independent experiments are shown. *p

    Article Snippet: MtDNA was isolated from mouse liver by using Mitochondrial DNA Isolation Kit (abcam, ab65321).

    Techniques:

    Mitochondrial DAMP pathway does not affect neuronal cell death. ( a , b ) Effect of mitochondria isolated from HT22 cells on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to mitochondria isolated from the indicated number of cells (( a ) representative flow cytometry plots; ( b ) quantification as % dead cells from total of n = 6 experiments). p = n.s. for all comparisons. Propidium iodide (PI) was detected in the PE channel and annexin V in the FITC channel. ( c , d ) Effect of mitochondrial DNA isolated from HT22 cells on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to mitochondrial DNA isolated from the indicated number of cells (( c ) representative flow cytometry plots; ( d ) quantification as % dead cells from total of n = 6 experiments). p = n.s. for all comparisons.

    Journal: Antioxidants

    Article Title: Neuroprotection after Hemorrhagic Stroke Depends on Cerebral Heme Oxygenase-1

    doi: 10.3390/antiox8100496

    Figure Lengend Snippet: Mitochondrial DAMP pathway does not affect neuronal cell death. ( a , b ) Effect of mitochondria isolated from HT22 cells on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to mitochondria isolated from the indicated number of cells (( a ) representative flow cytometry plots; ( b ) quantification as % dead cells from total of n = 6 experiments). p = n.s. for all comparisons. Propidium iodide (PI) was detected in the PE channel and annexin V in the FITC channel. ( c , d ) Effect of mitochondrial DNA isolated from HT22 cells on neuronal cell death in HT22 cells in vitro analyzed by flow cytometry after annexin V/PI staining. Cells were exposed to mitochondrial DNA isolated from the indicated number of cells (( c ) representative flow cytometry plots; ( d ) quantification as % dead cells from total of n = 6 experiments). p = n.s. for all comparisons.

    Article Snippet: However, incubation of HT22 cells with mitochondria isolated from neuronal cells ( a,b) or with mitochondrial DNA from the indicated number of HT22 cells ( c,d) did not directly induce neuronal cell death, suggesting that the damaging effect of DAMPs is indirect by activation of the innate immune response, namely, microglia cells within the brain that trigger a damaging inflammatory response.

    Techniques: Isolation, In Vitro, Flow Cytometry, Cytometry, Staining

    N-Myc overexpression increased mitochondrial biogenesis. ( a ) Whole cell lysates (WCL) from SH and SH-N-Myc cells were collected and used for western analysis with N-Myc antibodies that showed N-Myc was highly overexpressed in our model. ( b ) WCL were used to measure expression of the global mitochondrial regulators PGC1-a and TFAM. Both are upregulated in SH-N-Myc. ( c ) Cells at mid-logarithmic phase were stained with MitoTracker Green and measured by flow cytometry. A representative curve is shown. ( d ) A qPCR-based assay was used to measure mitochondrial DNA copy number using genomic DNA content as the control. Four separate experiments were performed with each cell line being measured at least in triplicate each time. Error bars show standard error of the experiments. P values: * P

    Journal: Cell Death Discovery

    Article Title: N-Myc overexpression increases cisplatin resistance in neuroblastoma via deregulation of mitochondrial dynamics

    doi: 10.1038/cddiscovery.2016.82

    Figure Lengend Snippet: N-Myc overexpression increased mitochondrial biogenesis. ( a ) Whole cell lysates (WCL) from SH and SH-N-Myc cells were collected and used for western analysis with N-Myc antibodies that showed N-Myc was highly overexpressed in our model. ( b ) WCL were used to measure expression of the global mitochondrial regulators PGC1-a and TFAM. Both are upregulated in SH-N-Myc. ( c ) Cells at mid-logarithmic phase were stained with MitoTracker Green and measured by flow cytometry. A representative curve is shown. ( d ) A qPCR-based assay was used to measure mitochondrial DNA copy number using genomic DNA content as the control. Four separate experiments were performed with each cell line being measured at least in triplicate each time. Error bars show standard error of the experiments. P values: * P

    Article Snippet: Mitochondrial DNA content Logarithmically growing cells were collected and used for quantification of mitochondrial to nuclear DNA content according to the protocol provided for the Human Mitochondrial DNA (mtDNA) Monitoring Primer Set (Takara Bio, Inc., Mountain View, CA, USA).

    Techniques: Over Expression, Western Blot, Expressing, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

    SFXN4 knockout attenuates mitochondrial respiration and affects the steady state of level of respiratory complex proteins. ( A ) Changes in oxygen consumption rate in response to treatment with indicated metabolic inhibitors in K562 (Scramble/SFXN4 KO) cells. Basal respiration, mitochondrial ATP production, maximal respiration and spare capacity were quantified in 8 replicate experiments (means and standard deviation). ( B ) The mean mitochondrial DNA copy number in K562 (Scramble/SFXN4 KO) cells from 3 replicate experiments. ( C ) Immunoblot showing the levels of labile subunits from each of the five mitochondrial respiratory complexes in K562 (Scramble/SFXN4 KO) cells. Means and standard deviations of quantified data from three independent experiments are shown under the blot. Images are representative of three independent experiments.

    Journal: Scientific Reports

    Article Title: Sideroflexin 4 affects Fe-S cluster biogenesis, iron metabolism, mitochondrial respiration and heme biosynthetic enzymes

    doi: 10.1038/s41598-019-55907-z

    Figure Lengend Snippet: SFXN4 knockout attenuates mitochondrial respiration and affects the steady state of level of respiratory complex proteins. ( A ) Changes in oxygen consumption rate in response to treatment with indicated metabolic inhibitors in K562 (Scramble/SFXN4 KO) cells. Basal respiration, mitochondrial ATP production, maximal respiration and spare capacity were quantified in 8 replicate experiments (means and standard deviation). ( B ) The mean mitochondrial DNA copy number in K562 (Scramble/SFXN4 KO) cells from 3 replicate experiments. ( C ) Immunoblot showing the levels of labile subunits from each of the five mitochondrial respiratory complexes in K562 (Scramble/SFXN4 KO) cells. Means and standard deviations of quantified data from three independent experiments are shown under the blot. Images are representative of three independent experiments.

    Article Snippet: Mitochondrial DNA copy number Mitochondrial DNA copy number was measured using Human Mitochondrial DNA (mtDNA) monitoring Primer Set (TaKaRa) according to the manufacturer’s instructions.

    Techniques: Knock-Out, Standard Deviation

    False-positive detection of bisulfite-resistant cytosines (brCs) in the negative control amplicons (NCAs) of mtDNA by bisulfite pyrosequencing. BrCs were detected in gel-purified or unpurified, bisulfite-converted PCR amplicons by pyrosequencing for 15 CpG sites: 5 in human ND1, 5 in human CYTB, 2 in mouse ND1, and 3 in mouse CYTB. The p -value was calculated using paired t-test (two-tails). The lines show mean ± 95% Confidence Intervals.

    Journal: PLoS ONE

    Article Title: Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

    doi: 10.1371/journal.pone.0192722

    Figure Lengend Snippet: False-positive detection of bisulfite-resistant cytosines (brCs) in the negative control amplicons (NCAs) of mtDNA by bisulfite pyrosequencing. BrCs were detected in gel-purified or unpurified, bisulfite-converted PCR amplicons by pyrosequencing for 15 CpG sites: 5 in human ND1, 5 in human CYTB, 2 in mouse ND1, and 3 in mouse CYTB. The p -value was calculated using paired t-test (two-tails). The lines show mean ± 95% Confidence Intervals.

    Article Snippet: Purity of human mtDNA was evaluated by qPCR detection of mtDNA markers (MT-ND1 and MT-ND5) and nuclear DNA markers (SLCO2B1 and SERPINA1) using Human mtDNA Monitoring Primer Set (Takara Bio).

    Techniques: Negative Control, Purification, Polymerase Chain Reaction

    Detection of bisulfite-resistant cytosines in purified, linearized human mtDNA by bisulfite pyrosequencing using converted template-selective (A9515) and unselective (hND1) sequencing primers. Ratios of brCs were determined by bisulfite pyrosequencing (Mean±SD, triplicated assays). (A) The A9515 sequencing primer, which was highly selective to bisulfite-converted DNA, interrogated three CpG sites (CpG #3–5) whereas non-selective sequencing primer hND1 interrogated all these CpG sites plus two additional CpG sites (CpG #1 and 2). (B) Positive control assay was performed using in vitro partially methylated NCAs templates. High CpG methylation levels at three CpG sites (CpG #3–5) were detected using A9515 sequencing primer (CpG sites #1 and #2 were out of the assay coverage using this sequencing primer). hND1 sequencing primer detected high CpG methylation at all five CpG sites (CpG #1–5).

    Journal: PLoS ONE

    Article Title: Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

    doi: 10.1371/journal.pone.0192722

    Figure Lengend Snippet: Detection of bisulfite-resistant cytosines in purified, linearized human mtDNA by bisulfite pyrosequencing using converted template-selective (A9515) and unselective (hND1) sequencing primers. Ratios of brCs were determined by bisulfite pyrosequencing (Mean±SD, triplicated assays). (A) The A9515 sequencing primer, which was highly selective to bisulfite-converted DNA, interrogated three CpG sites (CpG #3–5) whereas non-selective sequencing primer hND1 interrogated all these CpG sites plus two additional CpG sites (CpG #1 and 2). (B) Positive control assay was performed using in vitro partially methylated NCAs templates. High CpG methylation levels at three CpG sites (CpG #3–5) were detected using A9515 sequencing primer (CpG sites #1 and #2 were out of the assay coverage using this sequencing primer). hND1 sequencing primer detected high CpG methylation at all five CpG sites (CpG #1–5).

    Article Snippet: Purity of human mtDNA was evaluated by qPCR detection of mtDNA markers (MT-ND1 and MT-ND5) and nuclear DNA markers (SLCO2B1 and SERPINA1) using Human mtDNA Monitoring Primer Set (Takara Bio).

    Techniques: Purification, Sequencing, Positive Control Assay, In Vitro, Methylation, CpG Methylation Assay

    Enrichment of human and mouse mtDNA over nuclear DNA. Ratios of copy numbers of human (A) or mouse (B) mtDNA and nuclear DNA were determined by qPCR of mtDNA and nuclear DNA marker genes (Mean±SD, triplicated assays). Numbers show fold enrichment compared to total DNA.

    Journal: PLoS ONE

    Article Title: Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

    doi: 10.1371/journal.pone.0192722

    Figure Lengend Snippet: Enrichment of human and mouse mtDNA over nuclear DNA. Ratios of copy numbers of human (A) or mouse (B) mtDNA and nuclear DNA were determined by qPCR of mtDNA and nuclear DNA marker genes (Mean±SD, triplicated assays). Numbers show fold enrichment compared to total DNA.

    Article Snippet: Purity of human mtDNA was evaluated by qPCR detection of mtDNA markers (MT-ND1 and MT-ND5) and nuclear DNA markers (SLCO2B1 and SERPINA1) using Human mtDNA Monitoring Primer Set (Takara Bio).

    Techniques: Real-time Polymerase Chain Reaction, Marker

    Induction of mitophagy by FA-M-β-CyD. Notes: ( A ) Effects of FA-M-β-CyD on expression of PINK1 in KB cells after incubation with 5 mM M-β-CyDs for 2 h. The PINK1 protein level was detected by Western blotting. The representative blots were indicated (n=3). ( B ) Effects of FA-M-β-CyD on mtDNA copy number in KB cells after incubation with 5 mM M-β-CyDs for 2 h. The mtDNA was quantified by real-time PCR. Bar graphs represent mean ± SEM (n=3–4 per group). Significant difference with P

    Journal: International Journal of Nanomedicine

    Article Title: Induction of mitophagy-mediated antitumor activity with folate-appended methyl-β-cyclodextrin

    doi: 10.2147/IJN.S133482

    Figure Lengend Snippet: Induction of mitophagy by FA-M-β-CyD. Notes: ( A ) Effects of FA-M-β-CyD on expression of PINK1 in KB cells after incubation with 5 mM M-β-CyDs for 2 h. The PINK1 protein level was detected by Western blotting. The representative blots were indicated (n=3). ( B ) Effects of FA-M-β-CyD on mtDNA copy number in KB cells after incubation with 5 mM M-β-CyDs for 2 h. The mtDNA was quantified by real-time PCR. Bar graphs represent mean ± SEM (n=3–4 per group). Significant difference with P

    Article Snippet: After quantification of DNA content using Epoch microplate photometer (BioTek Instruments, Winooski, VT, USA), the mtDNA content was measured using real-time polymerase chain reaction (PCR) using Human mtDNA Monitoring Primer Set® (TaKaRa Bio, Tokyo, Japan) following the manufacturer’s instructions.

    Techniques: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction

    Spatial sampling of a Barrett's segment and associated oesophageal adenocarcinoma (OAC). (A) Overview of the opened resection specimen shows columnar metaplasia across the gastro-oesophageal junction (arrows) and a nodular OAC (arrowhead). The rectangular box indicates the longitudinal strip that was sampled. (B) H E-stained cryostat section (left) of the longitudinal strip across the gastro-oesophageal junction reveals columnar metaplasia of the distal oesophagus (arrows) and an OAC at the squamocolumnar junction (arrowhead). Submucosal gland complex (asterisk) confirms the oesophageal origin. Cytochrome c oxidase (CCO) staining of this longitudinal strip (right) shows several discontinuous epithelial patches that are CCO-deficient. The OAC is also CCO-deficient. (B) and (C) images are taken at same magnification (see scalebar). The boxed area is shown in detail in (C). (C) Overview and high-power photomicrographs of one CCO-deficient epithelial patch and the associated OAC. Note the mosaic spread of the CCO-deficient clone within the background mucosa. There are no architectural or cytological features of dysplasia. OAC shows atypical, cribriform glands, which penetrate the pre-existent muscularis mucosae. (D) Deep next-generation mitochondrial DNA (mtDNA) sequencing reveals unique mtDNA mutations within the CCO-deficient epithelial patch shown in (C). Barchart shows the variant allele fractions (VAFs) of the mutations in control (stroma) sample and in material from the clonal expansion, mtDNA mutations are indicated. The 16290C > T mutation was selected for further analysis. (E) Sanger resequencing shows that spatially distinct CCO-deficient epithelial patches and the OAC carry the same 16290C > T mtDNA mutation (see also table 1 ). CCO-proficient epithelium does not carry this genetic lineage marker. (F) Consecutive sections of neighbouring glands showing cardia-type metaplasia and intestinal metaplasia. Top left: H E staining shows absence of goblet cell differentiation in non-dysplastic cardia-type epithelium (marked by arrowhead), whereas the neighbouring intestinal metaplasia shows abundant goblet cells (marked by asterisk). Top right: clonal loss of CCO activity in cardia-type epithelium (marked by arrowhead). Bottom left: CDX2 staining confirms absence of intestinalisation in CCO-deficient cardia-type metaplasia. Strong nuclear labelling is seen in neighbouring intestinal metaplasia (marked by arrowhead). Bottom right: low proliferative activity as shown by Ki67 proliferation marker stain, consistent with morphological absence of dysplasia. Arrowhead points to positive nuclear labelling.

    Journal: Gut

    Article Title: Evolution of oesophageal adenocarcinoma from metaplastic columnar epithelium without goblet cells in Barrett's oesophagus

    doi: 10.1136/gutjnl-2015-310748

    Figure Lengend Snippet: Spatial sampling of a Barrett's segment and associated oesophageal adenocarcinoma (OAC). (A) Overview of the opened resection specimen shows columnar metaplasia across the gastro-oesophageal junction (arrows) and a nodular OAC (arrowhead). The rectangular box indicates the longitudinal strip that was sampled. (B) H E-stained cryostat section (left) of the longitudinal strip across the gastro-oesophageal junction reveals columnar metaplasia of the distal oesophagus (arrows) and an OAC at the squamocolumnar junction (arrowhead). Submucosal gland complex (asterisk) confirms the oesophageal origin. Cytochrome c oxidase (CCO) staining of this longitudinal strip (right) shows several discontinuous epithelial patches that are CCO-deficient. The OAC is also CCO-deficient. (B) and (C) images are taken at same magnification (see scalebar). The boxed area is shown in detail in (C). (C) Overview and high-power photomicrographs of one CCO-deficient epithelial patch and the associated OAC. Note the mosaic spread of the CCO-deficient clone within the background mucosa. There are no architectural or cytological features of dysplasia. OAC shows atypical, cribriform glands, which penetrate the pre-existent muscularis mucosae. (D) Deep next-generation mitochondrial DNA (mtDNA) sequencing reveals unique mtDNA mutations within the CCO-deficient epithelial patch shown in (C). Barchart shows the variant allele fractions (VAFs) of the mutations in control (stroma) sample and in material from the clonal expansion, mtDNA mutations are indicated. The 16290C > T mutation was selected for further analysis. (E) Sanger resequencing shows that spatially distinct CCO-deficient epithelial patches and the OAC carry the same 16290C > T mtDNA mutation (see also table 1 ). CCO-proficient epithelium does not carry this genetic lineage marker. (F) Consecutive sections of neighbouring glands showing cardia-type metaplasia and intestinal metaplasia. Top left: H E staining shows absence of goblet cell differentiation in non-dysplastic cardia-type epithelium (marked by arrowhead), whereas the neighbouring intestinal metaplasia shows abundant goblet cells (marked by asterisk). Top right: clonal loss of CCO activity in cardia-type epithelium (marked by arrowhead). Bottom left: CDX2 staining confirms absence of intestinalisation in CCO-deficient cardia-type metaplasia. Strong nuclear labelling is seen in neighbouring intestinal metaplasia (marked by arrowhead). Bottom right: low proliferative activity as shown by Ki67 proliferation marker stain, consistent with morphological absence of dysplasia. Arrowhead points to positive nuclear labelling.

    Article Snippet: Mitochondrial DNA (mtDNA) was amplified in two overlapping fragments using high-fidelity Takara LA Taq (Clontech, Saint-Germain-en-Laye, France); all reactions were performed in duplicate.

    Techniques: Sampling, Stripping Membranes, Staining, Sequencing, Variant Assay, Mutagenesis, Marker, Cell Differentiation, Activity Assay

    Quantification of mtDNA enrichment by qPCR. A. Quantification after nuclear DNA removal using exonuclease V (ExoV) as indicated. B. Quantification after mtDNA amplification using MDA. The results are given for DNA untreated by exoV (No ExoV), or after two conditions of ExoV digestion as indicated. REPLI-g was performed using primers at a concentration of 0.2 µM or 1.0 µM.

    Journal: bioRxiv

    Article Title: A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

    doi: 10.1101/2019.12.20.884486

    Figure Lengend Snippet: Quantification of mtDNA enrichment by qPCR. A. Quantification after nuclear DNA removal using exonuclease V (ExoV) as indicated. B. Quantification after mtDNA amplification using MDA. The results are given for DNA untreated by exoV (No ExoV), or after two conditions of ExoV digestion as indicated. REPLI-g was performed using primers at a concentration of 0.2 µM or 1.0 µM.

    Article Snippet: Whole mitochondrial genome amplification (WMGA) We used the REPLI-g mitochondrial DNA kit (Qiagen) to perform WMGA according to the manufacturer’s instruction with the following modifications.

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Multiple Displacement Amplification, Concentration Assay

    Analysis of direct repeat (DR) motifs in mitochondrial genomes. Abundance, distribution and free energy of DRs in mtDNA and random DNA sequences ( n = 100) with the same base composition (R1) as corresponding mtDNA. (A) Frequency of DR pairs (≥6 bp) in the mtDNA of human, rhesus monkey, mouse and rat. The DR frequency is normalized with respect to the mtDNA length in each species. (B) The distribution of left and right DR sequences in the minor and major arcs of human mtDNA and the mean distribution of DRs in R1 random sequences. (C) DR sizes and DR free energies in human mtDNA and the corresponding R1 random sequences. The lower the free energy of a DR, i.e. the more negative, the more stable is the DNA duplex formed. (D) Distribution of free energies of DNA duplex formed by DRs (≥6 bp) in native mtDNA and random sequences (R1) of human, rhesus monkey, mouse and rat.

    Journal: PLoS ONE

    Article Title: Role of Direct Repeat and Stem-Loop Motifs in mtDNA Deletions: Cause or Coincidence?

    doi: 10.1371/journal.pone.0035271

    Figure Lengend Snippet: Analysis of direct repeat (DR) motifs in mitochondrial genomes. Abundance, distribution and free energy of DRs in mtDNA and random DNA sequences ( n = 100) with the same base composition (R1) as corresponding mtDNA. (A) Frequency of DR pairs (≥6 bp) in the mtDNA of human, rhesus monkey, mouse and rat. The DR frequency is normalized with respect to the mtDNA length in each species. (B) The distribution of left and right DR sequences in the minor and major arcs of human mtDNA and the mean distribution of DRs in R1 random sequences. (C) DR sizes and DR free energies in human mtDNA and the corresponding R1 random sequences. The lower the free energy of a DR, i.e. the more negative, the more stable is the DNA duplex formed. (D) Distribution of free energies of DNA duplex formed by DRs (≥6 bp) in native mtDNA and random sequences (R1) of human, rhesus monkey, mouse and rat.

    Article Snippet: Mitochondrial DNA and random DNA sequences The mitochondrial DNA (mtDNA) sequences used in this study were obtained from the National Centre for Biotechnology Information (NCBI) database.

    Techniques:

    Exclusion of mitochondrial DNA (mtDNA) mutations occurring in the germline or in early embryogenesis. ( A ) Somatic mtDNA frequency (mtDNA mutations present in colon only) measured by NGS. There was a significantly higher mutation frequency in the subjects > 70 years (P = 0.0351, unpaired t-test). ( B ) Percentage of synonymous/polymorphic variants and non-synonymous mtDNA mutations which were of germline or early embryological origin compared to those which were somatic in adults

    Journal: PLoS Genetics

    Article Title: Clonal Expansion of Early to Mid-Life Mitochondrial DNA Point Mutations Drives Mitochondrial Dysfunction during Human Ageing

    doi: 10.1371/journal.pgen.1004620

    Figure Lengend Snippet: Exclusion of mitochondrial DNA (mtDNA) mutations occurring in the germline or in early embryogenesis. ( A ) Somatic mtDNA frequency (mtDNA mutations present in colon only) measured by NGS. There was a significantly higher mutation frequency in the subjects > 70 years (P = 0.0351, unpaired t-test). ( B ) Percentage of synonymous/polymorphic variants and non-synonymous mtDNA mutations which were of germline or early embryological origin compared to those which were somatic in adults

    Article Snippet: Mitochondrial DNA (mtDNA) mutations detected by Ion Torrent Next Generation Sequencing in human buccal epithelium. (PDF) Click here for additional data file.

    Techniques: Next-Generation Sequencing, Mutagenesis

    Analysis of mitochondrial DNA point mutation frequency with age by Random Mutation Capture (RMC). ( A ) Schematic diagram describing the RMC methodology. ( i ) Schematic diagram of the structure of the human colorectal crypt. ( ii ) Schematic diagram showing mtDNA isolation. Colonoscopic biopsies are homogenized and the mitochondrial fraction isolated by differential centrifugation. MtDNA is then prepared by phenol/chloroform extraction and quantified using real-time PCR (standard curve method). ( iii ) MtDNA is digested for 10 hours with Taq1α . PCR is then carried out over the restriction site. Only molecules with mutations in the restriction site are able to successfully amplify. ( iv ) Agarose gel showing PCR products from a typical RMC run. Each reaction contained ∼10000 target base pairs. 488 base pair bands show amplified mutated molecules (wells 4,6,13,16 and 20). The wild-type control well (Wt) shows complete digestion of wild-type DNA following PCR. ( v ) Example electropherograms showing mutations (asterisks) within the restriction site (highlighted in blue). ( B ) Frequency of all RMC detected mtDNA mutations in human colorectal mucosa (n = 207). There was no correlation between mtDNA mutation frequency and age (Pearson correlation = 0.127 (P = 0.07)). ( C ) Data from ( B ) presented on a log 10 scale to show the spread of the data. Note that the zero values cannot be displayed in this way therefore n = 175. ( D ) Frequency of all mtDNA mutations detected in human colonic mucosa, grouped by decade. Subjects were grouped as follows, 17–26 (n = 12), 27–36 (n = 19), 37–46 (n = 58), 47–56 (n = 51), 57–66 (n = 43), 67–77 (n = 23). There were no significant differences between any of the groups (P = 0.343, One Way ANOVA).

    Journal: PLoS Genetics

    Article Title: Clonal Expansion of Early to Mid-Life Mitochondrial DNA Point Mutations Drives Mitochondrial Dysfunction during Human Ageing

    doi: 10.1371/journal.pgen.1004620

    Figure Lengend Snippet: Analysis of mitochondrial DNA point mutation frequency with age by Random Mutation Capture (RMC). ( A ) Schematic diagram describing the RMC methodology. ( i ) Schematic diagram of the structure of the human colorectal crypt. ( ii ) Schematic diagram showing mtDNA isolation. Colonoscopic biopsies are homogenized and the mitochondrial fraction isolated by differential centrifugation. MtDNA is then prepared by phenol/chloroform extraction and quantified using real-time PCR (standard curve method). ( iii ) MtDNA is digested for 10 hours with Taq1α . PCR is then carried out over the restriction site. Only molecules with mutations in the restriction site are able to successfully amplify. ( iv ) Agarose gel showing PCR products from a typical RMC run. Each reaction contained ∼10000 target base pairs. 488 base pair bands show amplified mutated molecules (wells 4,6,13,16 and 20). The wild-type control well (Wt) shows complete digestion of wild-type DNA following PCR. ( v ) Example electropherograms showing mutations (asterisks) within the restriction site (highlighted in blue). ( B ) Frequency of all RMC detected mtDNA mutations in human colorectal mucosa (n = 207). There was no correlation between mtDNA mutation frequency and age (Pearson correlation = 0.127 (P = 0.07)). ( C ) Data from ( B ) presented on a log 10 scale to show the spread of the data. Note that the zero values cannot be displayed in this way therefore n = 175. ( D ) Frequency of all mtDNA mutations detected in human colonic mucosa, grouped by decade. Subjects were grouped as follows, 17–26 (n = 12), 27–36 (n = 19), 37–46 (n = 58), 47–56 (n = 51), 57–66 (n = 43), 67–77 (n = 23). There were no significant differences between any of the groups (P = 0.343, One Way ANOVA).

    Article Snippet: Mitochondrial DNA (mtDNA) mutations detected by Ion Torrent Next Generation Sequencing in human buccal epithelium. (PDF) Click here for additional data file.

    Techniques: Mutagenesis, Isolation, Centrifugation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification

    Mitochondrial DNA mutations in human colorectal epithelium of subjects below 26 years of age (n = 8) and over 70 years of age (n = 8) measured by Next Generation Sequencing (NGS). ( A ) MtDNA mutation frequency. There was a significantly higher mutation frequency in the subjects > 70 years (p = 0.0361, unpaired t-test). ( B ) Types of mutations detected by NGS frequency. There was no significant difference in the types of mutations detected in subjects

    Journal: PLoS Genetics

    Article Title: Clonal Expansion of Early to Mid-Life Mitochondrial DNA Point Mutations Drives Mitochondrial Dysfunction during Human Ageing

    doi: 10.1371/journal.pgen.1004620

    Figure Lengend Snippet: Mitochondrial DNA mutations in human colorectal epithelium of subjects below 26 years of age (n = 8) and over 70 years of age (n = 8) measured by Next Generation Sequencing (NGS). ( A ) MtDNA mutation frequency. There was a significantly higher mutation frequency in the subjects > 70 years (p = 0.0361, unpaired t-test). ( B ) Types of mutations detected by NGS frequency. There was no significant difference in the types of mutations detected in subjects

    Article Snippet: Mitochondrial DNA (mtDNA) mutations detected by Ion Torrent Next Generation Sequencing in human buccal epithelium. (PDF) Click here for additional data file.

    Techniques: Next-Generation Sequencing, Mutagenesis

    Mitochondrial BER deficient mice do not accumulate point mutations to mtDNA after five generations of consecutive breeding. ( A ) Breeding scheme to accumulate mutations into mtDNA and study germ line mutations. Homozygous Mutyh dMTS × Ogg1 dMTS female mice were bred with homo- or heterozygous Mutyh dMTS × Ogg1 dMTS male mice for five consecutive generations. To minimize the nuclear effects, heterozygote male mice were also used in the breedings. N1–N5 indicates the generations of breeding. ( B ) Mutation load of mtDNA with Illumina sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. The sequencing was carried out from purified mtDNA from liver. Data is quality filtered and minimum variant allele frequency is set to 0.5%. In unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. In total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (++ n = 6, pp n = 2, 10–13 week old) and gray circles indicate samples from homozygous Mutyh dMTS × Ogg1 dMTS mice (dd dd, n = 8, 10–15 week old). Horizontal lines represent means. C. Mutation profile of mtDNA with Illumina sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. Samples as in B. Horizontal lines represent means. For only quality filtered data see Supplementary Figure S3 .

    Journal: Nucleic Acids Research

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    doi: 10.1093/nar/gky456

    Figure Lengend Snippet: Mitochondrial BER deficient mice do not accumulate point mutations to mtDNA after five generations of consecutive breeding. ( A ) Breeding scheme to accumulate mutations into mtDNA and study germ line mutations. Homozygous Mutyh dMTS × Ogg1 dMTS female mice were bred with homo- or heterozygous Mutyh dMTS × Ogg1 dMTS male mice for five consecutive generations. To minimize the nuclear effects, heterozygote male mice were also used in the breedings. N1–N5 indicates the generations of breeding. ( B ) Mutation load of mtDNA with Illumina sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. The sequencing was carried out from purified mtDNA from liver. Data is quality filtered and minimum variant allele frequency is set to 0.5%. In unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. In total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (++ n = 6, pp n = 2, 10–13 week old) and gray circles indicate samples from homozygous Mutyh dMTS × Ogg1 dMTS mice (dd dd, n = 8, 10–15 week old). Horizontal lines represent means. C. Mutation profile of mtDNA with Illumina sequencing from Mutyh dMTS × Ogg1 dMTS mice after five generations of consecutive breeding. Samples as in B. Horizontal lines represent means. For only quality filtered data see Supplementary Figure S3 .

    Article Snippet: To increase the depth of mutation analysis and to expand the coverage to the entire mtDNA, we used Illumina sequencing of purified mtDNA to assess the mutation load.

    Techniques: Mouse Assay, Mutagenesis, Sequencing, Purification, Variant Assay

    Mitochodrial BER deficient mice do not accumulate point mutations to mtDNA even in the presence of increased oxidative stress. ( A ) Mutation load of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Data is quality filtered and minimum variant allele frequency is set to 0.5%. For the unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (pp n = 4 or ++ n = 3, 8–12 week old), light gray circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4 or +p dd n = 2 or +p cre+ dd n = 1, 8–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 7, 9–10 week old). Horizontal lines represent means, one-way ANOVA, Tukey's multiple comparison test. ( B ) Mutation profile of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Samples as in A. Horizontal lines represent mean. For only quality-filtered data see Supplementary Figure S4 .

    Journal: Nucleic Acids Research

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    doi: 10.1093/nar/gky456

    Figure Lengend Snippet: Mitochodrial BER deficient mice do not accumulate point mutations to mtDNA even in the presence of increased oxidative stress. ( A ) Mutation load of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Data is quality filtered and minimum variant allele frequency is set to 0.5%. For the unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (pp n = 4 or ++ n = 3, 8–12 week old), light gray circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4 or +p dd n = 2 or +p cre+ dd n = 1, 8–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 7, 9–10 week old). Horizontal lines represent means, one-way ANOVA, Tukey's multiple comparison test. ( B ) Mutation profile of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Samples as in A. Horizontal lines represent mean. For only quality-filtered data see Supplementary Figure S4 .

    Article Snippet: To increase the depth of mutation analysis and to expand the coverage to the entire mtDNA, we used Illumina sequencing of purified mtDNA to assess the mutation load.

    Techniques: Mouse Assay, Mutagenesis, Sequencing, Purification, Variant Assay

    Mitochondrial BER deficient mice do not accumulate point mutations of mtRNA even in the presence of increased oxidative stress. ( A ) Mutation load of mtRNA from Sod2 loxP x Ckmm cre mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. For the unique mutation load each specific mutation is counted only once, reflecting how many times a mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre mice from heart. White circles indicate samples from controls (+p n = 1 pp n = 2, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre n = 3, 10–11 week old). ( B ) Mutation load of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. White circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 4, 9–10 week old). * P

    Journal: Nucleic Acids Research

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    doi: 10.1093/nar/gky456

    Figure Lengend Snippet: Mitochondrial BER deficient mice do not accumulate point mutations of mtRNA even in the presence of increased oxidative stress. ( A ) Mutation load of mtRNA from Sod2 loxP x Ckmm cre mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. For the unique mutation load each specific mutation is counted only once, reflecting how many times a mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre mice from heart. White circles indicate samples from controls (+p n = 1 pp n = 2, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre n = 3, 10–11 week old). ( B ) Mutation load of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. White circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 4, 9–10 week old). * P

    Article Snippet: To increase the depth of mutation analysis and to expand the coverage to the entire mtDNA, we used Illumina sequencing of purified mtDNA to assess the mutation load.

    Techniques: Mouse Assay, Mutagenesis, Sequencing, Variant Assay