Journal: bioRxiv

Article Title: Diabetic conditions induce intolerance to accumulation of pathogenic mitochondrial DNAs

doi: 10.1101/790956

Figure Lengend Snippet: Changes in mitochondrial biogenesis in db/db-mtΔ mice and wt/wt-mtΔ mice. In (A) and (B), the upper graphs show plots of all values and the bottom ones show averaged values of four groups carrying 0%, 3% to 48%, 51% to 71%, and 73% to 87% ΔmtDNA in the cardiac muscle tissues. Statistical analysis was performed by using a t -test for comparison with wt/wt-mtΔ mice in each group. (A) Expression levels of PGC1α, Tfam, and mt-Co2 mRNAs in cardiac muscle tissues of wt/wt-mtΔ mice carrying 0% to 86% ΔmtDNA (open squares and bars) and db/db-mtΔ mice carrying 0% to 87% ΔmtDNA (solid squares and bars). Levels of PGC1α, Tfam, and mt-Co2 mRNAs in cardiac muscle tissues from db/db-mtΔ mice were significantly lower than in those from wt/wt-mtΔ mice. (B) Changes in mtDNA copy numbers in cardiac muscle tissues of wt/wt-mtΔ mice (open squares and bars) and db/db-mtΔ mice (solid squares and bars). Compared with those in wt/wt-mtΔ mice, mtDNA copy numbers in the cardiac muscle tissues from db/db-mtΔ mice were significantly lower, except for a case of 3% to 48% ΔmtDNA. In the bottom graphs, all values are means ± SD. *P

Article Snippet: Gene expression levels were assayed by using a Taqman gene expression assay with mouse PGC1α (Mm 01208835_m1) and mouse Tfam (Mm 00447485_m1) in real-time PCR analysis.

Techniques: Mouse Assay, Expressing

Journal: bioRxiv

Article Title: Diabetic conditions induce intolerance to accumulation of pathogenic mitochondrial DNAs

doi: 10.1101/790956

Figure Lengend Snippet: Characterization of human cybrids carrying mtDNA with the A3243G mutation in diabetic culture conditions. In this experiment, two human cybrids obtained by the repopulation of mtDNA-free HeLa cells with only wild-type mtDNA (HeLa-WT cybrids) or with both wild-type mtDNA and pathogenic A3243G mtDNA (HeLa-A3243G cybrids) were used. The HeLa-A3243G cybrids carried 89% A3243G mtDNA. The cybrids were cultured for 7 days with normal medium or high-glucose medium (representing diabetic culture conditions). In (B) to (E), open and solid bars indicate normal and diabetic culture conditions, respectively. (A) Cytochemical observations of COX and SDH activities. Cybrids that were normal or deficient in mitochondrial respiration activity were visualized as brown (COX+/SDH+) or blue (COX–/SDH+), respectively. The number of blue HeLa-A3243G cybrids increased, but that of HeLa-WT cybrids did not, when the cybrids were cultured in high-glucose medium. Scale bar, 50 µm. (B) Quantification of number of cybrids showing mitochondrial respiration defects. In comparison with culture in normal medium, the proportion of HeLa-A3243G cybrids visualized as blue (COX-/SDH+) increased significantly in culture with high-glucose medium. (C) Biochemical analysis for COX activity. The biochemical COX activity of HeLa-A3243G cybrids was half that of HeLa-WT cybrids in normal culture condition, because the HeLa-A3243G cybrids harbored large amounts of A3243G mtDNA (89% load). Culture in diabetic conditions further decreased the biochemical COX activity in HeLa-A3243G cybrids. (D) Levels of PGC1α, Tfam, and mt-Co2 mRNAs in HeLa-wt and HeLa-A3243G cybrids. HeLa-A3243G cybrids showed significantly lower levels of PGC1α, Tfam, and mt-Co2 mRNAs in diabetic culture conditions than in normal culture conditions. (E) Copy numbers of mtDNA in HeLa-wt and HeLa-A3243G cybrids. HeLa-A3243G cybrids in diabetic culture had significantly lower mtDNA copy numbers than in normal culture. All values are means ± 1 SD. *P

Article Snippet: Gene expression levels were assayed by using a Taqman gene expression assay with mouse PGC1α (Mm 01208835_m1) and mouse Tfam (Mm 00447485_m1) in real-time PCR analysis.

Techniques: Mutagenesis, Cell Culture, Activity Assay

Journal: PLoS Biology

Article Title: Jagged1/Notch2 controls kidney fibrosis via Tfam-mediated metabolic reprogramming

doi: 10.1371/journal.pbio.2005233

Figure Lengend Snippet: Tfam rescues TGF-β1/Jag1/Notch2-induced metabolic defect and downstream profibrotic changes. (A) Tfam expression level in NRK-52E cells treated with TGF-β1 for 24 h. Data are represented as mean ± SD. ** P

Article Snippet: Proteins were resolved on 8%–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with antibodies as below: JAG1 (Abcam #ab109536; 1:1000), NOTCH2 (Abcam #ab8926; 1:500), TFAM (Millipore #ABS2083; 1:1000), and β actin (Abcam #ab8226; 1:5000).

Techniques: Expressing

Journal: PLoS Biology

Article Title: Jagged1/Notch2 controls kidney fibrosis via Tfam-mediated metabolic reprogramming

doi: 10.1371/journal.pbio.2005233

Figure Lengend Snippet: Tubule-specific Jag1 / Notch2 signaling plays a key role in kidney fibrosis development by regulating metabolism via Tfam . TEC, tubular epithelial cells; Tfam, mitochondrial transcription factor A.

Article Snippet: Proteins were resolved on 8%–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with antibodies as below: JAG1 (Abcam #ab109536; 1:1000), NOTCH2 (Abcam #ab8926; 1:500), TFAM (Millipore #ABS2083; 1:1000), and β actin (Abcam #ab8226; 1:5000).

Techniques:

Journal: Molecular Medicine Reports

Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway

doi: 10.3892/mmr.2019.9853

Figure Lengend Snippet: NaN 3 induces mitochondria-mediated apoptosis through the expression of Pgc-1α-associated proteins in PC12 cells. (A) Expression levels of Pgc-1α, Nrf-1, Nrf-2, Tfam and Cox IV detected by western blot analysis. (B) Expression levels of procaspase-3, Bax, Bcl-2 and cyt-c detected by western blot analysis. (C) Expression levels of pan-calcineurin A, CaMKII, p-CaMKII, p38 MAPK, p-p38 MAPK, Erk1/2 and p-Erk1/2 detected by western blot analysis. β-actin and GAPDH were used as the internal control. Band intensity ratios for each group are presented as mean ± standard deviation (n=3). *P

Article Snippet: Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight.

Techniques: Expressing, Pyrolysis Gas Chromatography, Western Blot, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Irisin Improves Autophagy of Aged Hepatocytes via Increasing Telomerase Activity in Liver Injury

doi: 10.1155/2020/6946037

Figure Lengend Snippet: The old rats showed more a serious decrease of irisin expression, telomerase activity, autophagy ability, and mitochondrial function than young rats during hepatic IR. Partial (70%) liver arterial/portal venous blood was interrupted for 40 and 60 minutes in 3-month- and 22-month-old rats. Liver samples were harvested at 24 h after reperfusion. (a, b) Western blot analysis of liver irisin expression. (c) Serum irisin levels. (d, e) Western blot analysis of liver telomerase reverse transcriptase (TERT) expression. (f–h) qPCR analysis of liver TERT , TERC , and TERF1 expression. (i) TEM analysis. The red arrow indicates autophagosomes. (j–l) Western blot analysis of liver LC3B and P62 expression. (m–o) Western blot analysis of liver PGC1 α and TFAM expression. (p–r) qPCR analysis of liver PGC1α , TFAM , and NRF1 expression. n = 6; mean ± SEM; ∗ P

Article Snippet: The primary rabbit anti-irisin antibody (1 : 1000 dilution, ab174833, Abcam, USA); rabbit anti-TERT antibody (1 : 1000 dilution, ab32020, Abcam, USA); rabbit anti-LC3B antibody (1 : 1,000 dilution, 3868, Cell Signaling Technology, USA); rabbit anti-P62 antibody (1 : 1,000 dilution, 5114, Cell Signaling Technology, USA); rabbit anti-PGC1α antibody (ab54481, Abcam, USA, 1 : 200 dilution); rabbit anti-TFAM antibody (ab131607, Abcam, USA, 1 : 200 dilution); rabbit anti-P-p38 antibody (1 : 1,000 dilution, 4511s, Cell Signaling Technology, USA); rabbit anti-P-ERK antibody (1 : 1,000 dilution, 4370s, Cell Signaling Technology, USA); rabbit anti-P-JNK antibody (1 : 1,000 dilution, 4668s, Cell Signaling Technology, USA); MAPK family antibody sampler kit (1 : 1,000 dilution, 9926, Cell Signaling Technology, USA); and rabbit anti-β -actin antibody (1 : 1000 dilution, 4967, Cell Signaling Technology, USA) were incubated overnight at 4°C on a shaker.

Techniques: Expressing, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Transmission Electron Microscopy

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Irisin Improves Autophagy of Aged Hepatocytes via Increasing Telomerase Activity in Liver Injury

doi: 10.1155/2020/6946037

Figure Lengend Snippet: Irisin improved telomerase activity, autophagy, and mitochondrial function in old rats after hepatic IR. Partial (70%) liver arterial/portal venous blood was interrupted for 40 minutes in 3-month- and 22-month-old rats. Irisin was administrated in old rats (iv. 250 μ g/kg, single dose) at the beginning of reperfusion. Irisin-neutralizing antibody was administrated in young rats (iv. 50 μ g/kg, single dose) at 24 h before hepatic IR. Liver samples were harvested at 24 h after reperfusion. (a–c) Western blot analysis of liver irisin and TERT expression. (d–f) qPCR analysis of liver TERF1 , TERC , and TERT expression in old rats. (g) TEM analysis. The red arrow indicates autophagosomes. (h–j) Western blot analysis of liver LC3B and P62 expression. (k–m) Western blot analysis of liver PGC1 α and TFAM expression. (n–p) qPCR analysis of liver PGC1α , TFAM , and NRF1 expression; n = 6; mean ± SEM; ∗ P

Article Snippet: The primary rabbit anti-irisin antibody (1 : 1000 dilution, ab174833, Abcam, USA); rabbit anti-TERT antibody (1 : 1000 dilution, ab32020, Abcam, USA); rabbit anti-LC3B antibody (1 : 1,000 dilution, 3868, Cell Signaling Technology, USA); rabbit anti-P62 antibody (1 : 1,000 dilution, 5114, Cell Signaling Technology, USA); rabbit anti-PGC1α antibody (ab54481, Abcam, USA, 1 : 200 dilution); rabbit anti-TFAM antibody (ab131607, Abcam, USA, 1 : 200 dilution); rabbit anti-P-p38 antibody (1 : 1,000 dilution, 4511s, Cell Signaling Technology, USA); rabbit anti-P-ERK antibody (1 : 1,000 dilution, 4370s, Cell Signaling Technology, USA); rabbit anti-P-JNK antibody (1 : 1,000 dilution, 4668s, Cell Signaling Technology, USA); MAPK family antibody sampler kit (1 : 1,000 dilution, 9926, Cell Signaling Technology, USA); and rabbit anti-β -actin antibody (1 : 1000 dilution, 4967, Cell Signaling Technology, USA) were incubated overnight at 4°C on a shaker.

Techniques: Activity Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transmission Electron Microscopy

Journal: Nature Communications

Article Title: Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts

doi: 10.1038/s41467-018-05077-9

Figure Lengend Snippet: Exosomes shuttle mitochondrial proteins. a Differential ultracentrifugation protocol for isolating and purifying exosomes from cell culture supernatants. b Size distribution analysis by Nanoparticle Tracking Analysis (NTA) of purified EVs from primary human T lymphoblasts. c Gene ontology (GO) cellular component analysis of peptides identified in T cell EVs. d Western blot analysis of mitochondrial proteins in EVs obtained from the culture supernatant of primary human T lymphoblasts with or without treatment with phorbol myristate acetate (PMA) plus ionomycin. Cells and EVs were blotted for proteins associated with mtDNA (TFAM and ATAD3), for proteins located in the inner mitochondrial membrane (COX1 and Cytochrome C), the outer mitochondrial membrane (VDAC1), the mitochondrial matrix (mitochondrial manganese superoxide dismutase, MnSOD), and for the exosome markers TSG101 and CD81. e Western blot analysis of ATAD3, TFAM, and the exosome markers CD81 and TSG101 in sucrose fractions. EVs obtained from the culture supernatant of human T lymphoblasts were laid on a discontinuous sucrose gradient and floated by overnight centrifugation. Gradient fractions were collected and analyzed by immunoblot to reveal the distribution of mtDNA-binding proteins and exosomal proteins in the sucrose fractions from lower to higher sucrose density (left to right). Gels shown are representative out of three independent experiments

Article Snippet: Antibodies and reagents The following antibodies were used for Western blotting: mouse anti-human CD63 (Calbiochem, OP171; 1:1000); mouse 5A6 anti-human CD81 (Santa Cruz, sc-23962; 1:500); mouse anti-human TSG101 (Abcam, ab83; 1:1000); mouse anti-GFP (Clontech, 632381; 1:2000); anti-TFAM (Abcam, ab47517; 1:500), anti-HRS (Abcam, ab72053; 1:500), anti-COX1 (Human Complex IV subunit I, Invitrogen, 459600; 1:1000), anti-VDAC1 (Abcam, ab14734; 1:1000), and anti-Cyt C (Abcam, ab110325; 1:1000), anti-LC3 (Cell Signaling, 2775 s; 1:1000); anti-MnSOD (Enzo Life Sciences, ADI-SOD-110; 1:1000), anti-ATAD3 (Abnova, H00055210-D01), anti-Tubulin (Sigma, T6199: 1:2000), anti-nSMase2 (RD System, MAB7184;1:1000) and anti-Rab27a (RD System, AF7245; 1:1000).

Techniques: Cell Culture, Purification, Western Blot, Centrifugation, Binding Assay

Journal: Oncotarget

Article Title: p53 increase mitochondrial copy number via up-regulation of mitochondrial transcription factor A in colorectal cancer

doi: 10.18632/oncotarget.12514

Figure Lengend Snippet: p53 expression following alteration of TFAM expression Western blot for determination of p53 and TFAM expression in cell lines treated with NC- or TFAM-siRNA A. and Empty- or TFAM-pEnter B. compared with control. Immunofluorescent staining of p53, TFAM and DAPI in cell lines with different treatment, compared with control C.

Article Snippet: Then, the section incubation was performed with p53 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TFAM antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, for assessment of p53 and TFAM, respectively.

Techniques: Expressing, Western Blot, Staining

Journal: Oncotarget

Article Title: p53 increase mitochondrial copy number via up-regulation of mitochondrial transcription factor A in colorectal cancer

doi: 10.18632/oncotarget.12514

Figure Lengend Snippet: Immunofluorescent staining of mitochondria and relative mtDNA copy number Immunofluorescent staining of mitochondria for cells treated with NC-, p53- or TFAM-siRNA, Empty-, p53- or TFAM-pEnter and pifithrin-α A , B , C. as well as RKO and RKO-E6 D. Relative mtDNA copy number of cells treated with NC-, p53- or TFAM-siRNA, Empty-, p53- or TFAM-pEnter and pifithrin-α expressed as percentage of control E , F , G , H , I. as well as RKO-E6 expressed as percentage of RKO J.

Article Snippet: Then, the section incubation was performed with p53 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TFAM antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, for assessment of p53 and TFAM, respectively.

Techniques: Staining

Journal: Oncotarget

Article Title: p53 increase mitochondrial copy number via up-regulation of mitochondrial transcription factor A in colorectal cancer

doi: 10.18632/oncotarget.12514

Figure Lengend Snippet: Association of p53 A. or TFAM B. expression and 5-year survival in colon adenocarcinoma tissues.

Article Snippet: Then, the section incubation was performed with p53 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TFAM antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, for assessment of p53 and TFAM, respectively.

Techniques: Expressing

Journal: Oncotarget

Article Title: p53 increase mitochondrial copy number via up-regulation of mitochondrial transcription factor A in colorectal cancer

doi: 10.18632/oncotarget.12514

Figure Lengend Snippet: p53 and TFAM expression in different colorectal cancer cell lines Western blot for determination of p53 and TFAM expression in cell lines of p53 wild type A. and p53 mutation type B. Quantitative analysis of p53 and TFAM expression comparing p53 wild type with p53 mutation type C. Quantitative analysis of p53 and TFAM expression in individual cell lines D. The linear correlation between p53 and TFAM expression in p53 wild type and p53 mutation type cell lines E. Immunofluorescent staining of p53, TFAM and DAPI in RKO and RKO-E6 cell lines F.

Article Snippet: Then, the section incubation was performed with p53 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TFAM antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, for assessment of p53 and TFAM, respectively.

Techniques: Expressing, Western Blot, Mutagenesis, Staining

Journal: Oncotarget

Article Title: p53 increase mitochondrial copy number via up-regulation of mitochondrial transcription factor A in colorectal cancer

doi: 10.18632/oncotarget.12514

Figure Lengend Snippet: TFAM expression following alteration of p53 expression Western blot for determination of p53 and TFAM expression in cell lines treated with NC- or p53-siRNA A. Empty- or p53-pEnter B. and pifithrin-α C. compared with control. Immunofluorescent staining of p53, TFAM and DAPI in cell lines with different treatment, compared with control D.

Article Snippet: Then, the section incubation was performed with p53 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TFAM antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, for assessment of p53 and TFAM, respectively.

Techniques: Expressing, Western Blot, Staining

Journal: Oncotarget

Article Title: p53 increase mitochondrial copy number via up-regulation of mitochondrial transcription factor A in colorectal cancer

doi: 10.18632/oncotarget.12514

Figure Lengend Snippet: TFAM promoter region-driven luciferase expression Relative luciferase activities were shown after different transfection patterns. pGL3-Basic: a firefly luciferase reporter vector; pGL4.75: a Renilla luciferase vector, working as an internal control; pGL3-Basic-TFAM: pGL3-Basic construct with TFAM promoter region (−1486 to +185 of TFAM gene); pCDNA3.1-p53: a plasmid which could highly express p53 after transfection.

Article Snippet: Then, the section incubation was performed with p53 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TFAM antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, for assessment of p53 and TFAM, respectively.

Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation, Construct

Journal: Oncotarget

Article Title: p53 increase mitochondrial copy number via up-regulation of mitochondrial transcription factor A in colorectal cancer

doi: 10.18632/oncotarget.12514

Figure Lengend Snippet: p53 and TFAM expression in colon adenocarcinoma tissues Immunohistochemistry of p53 and TFAM for colon adenocarcinoma and corresponding paracancerous tissues A. Scoring of p53 and TFAM staining according to staining index described in methods B , C. The linear correlation between p53 and TFAM expression in colon adenocarcinoma and corresponding paracancerous tissues D.

Article Snippet: Then, the section incubation was performed with p53 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TFAM antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, for assessment of p53 and TFAM, respectively.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Tetramethylpyrazine Analogue T-006 Exerts Neuroprotective Effects against 6-Hydroxydopamine-Induced Parkinson's Disease In Vitro and In Vivo

doi: 10.1155/2019/8169125

Figure Lengend Snippet: Effects of T-006 on the PKA/CREB/NRF1 signaling pathway and mitochondrial biogenesis in PC12 cells. PC12 cells were treated with T-006 (30 μ M) at the indicated time points, and the expression of nuclear PGC-1 α and NRF1 (a) and cytoplasmic TFAM (b) were detected by Western blot. (c) Cells were incubated with T-006 (30 μ M) as indicated, and the mtDNA content was quantified by quantitative real-time PCR. (d) The cells were treated with T-006 and harvested at the indicated times; the expression of phosphorylated CREB/total CREB was detected by Western blot analysis. (e) Cells were pretreated with or without PKA inhibitor H-89 (10 μ M) for 1 h and then treated with T-006 (30 μ M) for 30 min. The expression ratios of phosphorylated CREB/total CREB were detected by Western blot. Representative Western blot and data analysis of three independent experiments are shown. Data are represented as the means ± SD. ∗ P

Article Snippet: Antibodies against Nrf2, PGC-1α , NRF1, and TFAM were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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

Journal: The Journal of Biological Chemistry

Article Title: An unexpected role for the transcriptional coactivator isoform NT-PGC-1α in the regulation of mitochondrial respiration in brown adipocytes

doi: 10.1074/jbc.M117.778373

Figure Lengend Snippet: A proposed scheme of the transcriptional control of mitochondrial respiratory function by NT-PGC-1α in brown adipocytes. Cold-induced PGC-1α and NT-PGC-1α transcriptionally regulate mitochondrial respiratory function in brown adipocytes by stimulating the expression of nucDNA-encoded ETC genes and other mitochondrial genes, including TFAM. TFAM is subsequently imported to mitochondria and regulates mitochondrial DNA replication and transcription, leading to an increase in mtDNA-encoded ETC gene expression. Our findings suggest an additional mechanism by which NT-PGC-1α regulates mtDNA transcription within brown adipocyte mitochondria. Simultaneous localization of NT-PGC-1α in the nucleus and mitochondria may contribute to the coordinated regulation of nucDNA- and mtDNA-encoded ETC gene expression in response to cold.

Article Snippet: Antibodies used were as follows: monoclonal and polyclonal anti-PGC-1α , anti-UCP1 , anti-Tom20, anti-Lamin B1, anti-TFAM, and anti-LRP130 from Santa Cruz Biotechnology and anti-HA, anti-α-tubulin, anti-CoxIV, and anti-HSP60 from Abcam.

Techniques: Pyrolysis Gas Chromatography, Expressing

Journal: Cell Death Discovery

Article Title: Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns

doi: 10.1038/cddiscovery.2017.62

Figure Lengend Snippet: The levels of enrichment for POLGA, ESRRB and TFAM and DNA methylation at exon 2 of PolgA . ( a ) Levels of enrichment for PolgA in the O H in the D-loop region of the mitochondrial genome following ChIP using antibodies specific to POLGA and real-time PCR across the O H region; ( b ) levels of enrichment for ESRRB within the CpG island of PolgA as determined by ChIP using an anti-ESRRB antibody and real-time PCR across the region of interest in PolgA . ( c ) % Methylation of PolgA at exon 2 for CC9 mus , CC9 spretus , CC9 pahari and CC9 dunni cells. Pyrosequencing was performed for 11 CpGs found on mouse PolgA exon 2 (chr7: 79 464 669–79 464 845). Primers were designed using the mouse reference sequence from UCSC Genome Browser Dec. 2011 (GRCm38/mm10) Assembly. ( d ) Levels of enrichment for TFAM in the D-loop region of the mitochondrial genome following ChIP using an anti-TFAM antibody and real-time PCR. Data are expressed as mean±S.E.M. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test. * P

Article Snippet: Chromatin from 1×106 cells was immunoprecipitated with Protein G Dynabeads and an anti-POLGA antibody (G-6, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or anti-TFAM antibody (Santa Cruz Biotechnology, Inc.), or anti-ESRRB antibody (H6705, R & D Systems, Minneapolis, MN, USA).

Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Methylation, Sequencing

Journal: Scientific Reports

Article Title: Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition

doi: 10.1038/s41598-018-19930-w

Figure Lengend Snippet: The diagram illustrates how radiation enhanced mitochondrial biogenesis and metabolic hyperactivation results in the increased formazan formation per cell. Ionizing radiation leads to increased intracellular Ca 2+ (step 1), which further accumulates in mitochondria and activate mitochondrial biogenesis signaling (step 2). Activation of Ca 2+ dependent signaling results in PGC-1α over-expression (step 3), which further transactivates the over-expression of Tfam (step 4), which induces mitochondrial biogenesis (step 5). Enhanced mitochondrial mass buffers cytoplasmic Ca 2+ and turns in to metabolically hyperactive mitochondria (step 6).

Article Snippet: The membrane was then incubated in 4% milk (according to manufacture protocol) for 2 h followed by primary antibody incubation SDH-A (1:1000), PGC-1α (1:1000), TFAM (1:1000) from Cell signaling technology, β-Actin (1:5000) from Santa Cruz Biotechnology.

Techniques: Activation Assay, Pyrolysis Gas Chromatography, Over Expression, Metabolic Labelling

Journal: Nature Communications

Article Title: Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts

doi: 10.1038/s41467-018-05077-9

Figure Lengend Snippet: Transfer of mitochondrial components during immune cognate interactions. a Flow cytometry analysis of Raji B cells incubated overnight with exosomes from J77 T cells control or stably expressing mitoDsRed (Representative experiment, n = 3). b Up: RFLP analysis of the mitochondrial genomes of C57 C57 and C57 NZB mice. Total DNA is from C57 C57 T lymphocytes and C57 NZB BMDCs. C57 C57 haplotype includes a BamHI restriction site; enzyme digestion results in two smaller fragments of 414 pb and 250 pb. Digestion of PCR products from C57 NZB haplotype results in a single band of 664 pb (full length). Down, RFLP detection of exogenous mtDNA in C57 NZB DCs incubated overnight with increasing amounts of exosomes from C57 C57 CD4 + T lymphoblasts. 414 pb and 250 pb fragments appear in C57 NZB DCs upon addition of exosomes (lanes 1/3, 2/3, and 3/3). Chart: Intensity profile of the RFLP analysis; arrows indicate the C57 mtDNA haplotype. C57 C57 CD4 + lane displays a band of 664 pb which corresponds to an uncompleted digestion of the PCR product in the RFLP analysis and a high exposure of the image. Representative experiment ( n = 3). c Exosome and mitochondrial transfer to unpulsed or SEE-pulsed Raji B cells (CMAC) from J77 T cells expressing the exosomal protein CD63GFP, the mitochondria-targeted mitoYFP or the mtDNA-binding protein TFAM-DsRED. Dot plots, Cell populations after co-culture. Red boxes enclose Raji B cells that have acquired exosomal or mitochondrial fluorescent markers (percentage from total Raji cells). Graphs: Percentage of Raji B cells acquiring fluorescence upon IS formation from 3 to 5 independent experiments; mean, t -test * P -value

Article Snippet: Samples were blocked with goat blocking solution (Aurion) for 1 h and incubated with primary antibodies; anti-Tfam (1:50) (Cell Signalling) and anti-DNA (1:15) (Promega) overnight in blocking solution.

Techniques: Flow Cytometry, Cytometry, Incubation, Stable Transfection, Expressing, Mouse Assay, Polymerase Chain Reaction, Binding Assay, Co-Culture Assay, Fluorescence

Journal: PPAR Research

Article Title: Cardiomyocyte-Restricted Deletion of PPARβ/δ in PPARα-Null Mice Causes Impaired Mitochondrial Biogenesis and Defense, but No Further Depression of Myocardial Fatty Acid Oxidation

doi: 10.1155/2011/372854

Figure Lengend Snippet: Expression of key determinants of mitochondrial biogenesis and mitochondrial proteins. (a) Real-time PCR measurement of transcript levels of NRF-1, NRF2 (a and b subunits), PGC-1 α and -1 β , and TFAM on samples from TMCM, PPAR α −/− , TMPD, and TMPDPA hearts. (b) Western blotting analyses of relative protein levels of PGC-1 α and TFAM on samples of nuclear proteins extracted from ventricular tissues of TMCM, PPAR α −/− , TMPD, and TMPDPA mice. (c) Transcript expression of mitochondrial proteins on samples from TMCM, PPAR α −/− , TMPD, and TMPDPA hearts. (d) Protein expression of mitochondrial proteins on samples from TMCM, PPAR α −/− , TMPD, and TMPDPA hearts. (e) Transcript expression of mitochondrial fission and fusion proteins on samples from TMCM, PPAR α −/|− , TMPD, and TMPDPA hearts. (f) Protein expression of mitochondrial fission and fusion proteins on samples from TMCM, PPAR α −/− , TMPD, and TMPDPA hearts. (g) The mitochondrial DNA copy number on samples from TMCM, PPAR α −/− , TMPD, and TMPDPA hearts. * P

Article Snippet: All antibodies were purchased from commercial sources: PPARβ /δ , FABP, CPT1b, and Catalase (Abcam); Glut1, Glut4, PGC-1α , Cyt b, DRP1, Fis1, SOD2, and GAPDH (Santa Cruz Biotechnology); TFam (Aviva); Cyt C (Invitrogen); Mfn2, and pan-actin (Sigma Aldrich); SOD1 (Biodesign International).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Pyrolysis Gas Chromatography, Western Blot, Mouse Assay

Journal: PLoS Genetics

Article Title: PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix

doi: 10.1371/journal.pgen.1004279

Figure Lengend Snippet: PINK1 accumulates upon knockdown of Lon. (A) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc-tagged PINK1 genomic construct and a UAS-RNAi construct driven by elav-GAL4 . The RNAi constructs used were as follows: the non-specific control mCherry -RNAi (Control-R), Lon -RNAi1 (Lon-R1), and Lon -RNAi2 (Lon-R2). An anti-Myc antibody was used to detect PINK1 and an anti-Actin antibody was used as a protein loading control. (B) Densitometry of the PINK1-Myc bands from the indicated genotypes was performed using Fiji software. The PINK1-Myc band intensities were then normalized to their respective Actin loading controls, and these ratios were in turn normalized to the Control-R PINK1/Actin ratio. (C) Western blot analysis of Lon protease (Lon), Mitochondrial transcription factor A (TFAM), Heat shock protein 60 (Hsp60) and Actin in whole head homogenate from control and Lon deficient animals. (D) Quantification of the Lon band intensities from the indicated genotypes performed as described in B. (E) Quantification of the TFAM bands from the indicated genotypes performed as described in B. (F) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B. (G) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs. The graph shows the normalized fluorescence intensity of TMRE, an indicator of mitochondrial membrane potential, relative to age-matched control animals. Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples. The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity. All experiments described in this figure were repeated at least three times per genotype. Error bars represent the standard error of the mean (s.e.m.). * p

Article Snippet: Immunodetections with commercial antibodies were performed at the following concentrations: 1∶1000 mouse anti-Myc 9E10 (#M4439, Sigma), 1∶500 rabbit anti-LONP1 (#NBP1-81734, Novus Biologicals), 1∶500 rabbit anti-TFAM (#ab47548, Abcam), 1∶1000 rabbit anti-Hsp60 (#4870S, Cell Signaling Technology), 1∶1000 mouse anti-VDAC (#MSA03, MitoSciences), 1∶2000 mouse anti-OxPhos Complex V subunit β (#A21351, Molecular Probes/Life Technologies), 1∶1000 mouse anti-NDUFS3 (#ab14711, Abcam), 1∶3000 mouse anti-PDH (#MSP07, MitoSciences), 1∶50,000 mouse anti-Actin (#MAB1501, Chemicon/Bioscience Research Reagents).

Techniques: Western Blot, Transgenic Assay, Expressing, Construct, Software, Flow Cytometry, Cytometry, Fluorescence

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Alogliptin, a Dipeptidyl Peptidase‐4 Inhibitor, Alleviates Atrial Remodeling and Improves Mitochondrial Function and Biogenesis in Diabetic Rabbits

doi: 10.1161/JAHA.117.005945

Figure Lengend Snippet: Protein levels of mitochondrial biogenesis‐related proteins in left atrial tissue estimated by Western blot. A through F, adiponectin, AMP‐activated protein kinase (AMPK), phosphorylated AMPK (p‐ AMPK ), peroxisome proliferator–activated receptor‐γ coactivator 1α (PGC‐1α), nuclear respiratory factor‐1 (NRF‐1), and mitochondrial transcription factor A (Tfam) protein levels in the 3 groups. * P

Article Snippet: Total protein was fractionated by electrophoresis and transferred onto PVDF sheets (Millipore) and separately incubated with a specific antibody targeting adiponectin (1:2000; Abcom), AMPK (1:2000; Abcom), phosphorylated AMPK (1:2000; Abcom), PGC‐1α (1:1000; Abcom), transcription of nuclear respiratory factor‐1 (NRF‐1) (1:1000; Abcom), and mitochondrial transcription factor A (Tfam) (1:1000; Abcom), followed by incubation with appropriate peroxidase‐conjugated secondary antibodies.

Techniques: Western Blot, Pyrolysis Gas Chromatography

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Alogliptin, a Dipeptidyl Peptidase‐4 Inhibitor, Alleviates Atrial Remodeling and Improves Mitochondrial Function and Biogenesis in Diabetic Rabbits

doi: 10.1161/JAHA.117.005945

Figure Lengend Snippet: Peroxisome proliferator–activated receptor‐γ coactivator 1α (PGC‐1α), nuclear respiratory factor‐1 (NRF‐1), and mitochondrial transcription factor A (Tfam) mRNA expression estimated by real‐time polymerase chain reaction. ** P

Article Snippet: Total protein was fractionated by electrophoresis and transferred onto PVDF sheets (Millipore) and separately incubated with a specific antibody targeting adiponectin (1:2000; Abcom), AMPK (1:2000; Abcom), phosphorylated AMPK (1:2000; Abcom), PGC‐1α (1:1000; Abcom), transcription of nuclear respiratory factor‐1 (NRF‐1) (1:1000; Abcom), and mitochondrial transcription factor A (Tfam) (1:1000; Abcom), followed by incubation with appropriate peroxidase‐conjugated secondary antibodies.

Techniques: Pyrolysis Gas Chromatography, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: Increased hepatic mitochondrial FA oxidation reduces plasma and liver TG levels and is associated with regulation of UCPs and APOC-III in rats

doi: 10.1194/jlr.M074849

Figure Lengend Snippet: Organ weights and mitochondrial biomarkers in Wistar male rats given 100 mg/kg 1-triple TTA for 3 weeks compared with controls. A: Liver index [(liver weight/body weight) × 100]. B: mtDNA (NADH dehydrogenase 1) compared with nDNA (18S) relative to control. C: Heart weight. D: Epididymal WAT weight. E. Hepatic citrate synthase activity. F. Hepatic mRNA levels of PGC-1α ( Ppargc1a ) mitochondrial transcription factor A ( Tfam ) and cytochrome c ( Cycs ). Values are shown as mean ± SD (n = 4–8). Significant difference compared with controls was determined with Student’s t -test (* P ≤ 0.05, ** P ≤ 0.0001 **** P ≤ 0.0001).

Article Snippet: RT-PCR was performed on Sarstedt 384-well Multiply-PCR plates (Sarstedt Inc., Newton, NC,) using an ABI Prism 7900HT sequence detection system from Applied Biosystems with the software, SDS 2.3. mRNA levels of genes of interest were detected in 1× Taqman buffer by using the following probes and primers listed in alphabetical order: acetyl-CoA carboxylase (Acaca ) (Rn00573474_m1); medium-chain acyl-CoA dehydrogenase (Acadm ) (Rn00566390_m1); long-chain acyl-CoA dehydrogenase (Acadl ) (Rn00563121_m1); very-long-chain acyl-CoA dehydrogenase (Acadvl ) (Rn00563649_m1); 4-N -trimethylaminobutyraldehyde dehydrogenase (Aldh9a1 ) (Rn01491039_m1); ApoB (Rn01499054_m1); ApoC-II (Rn01764530_g1); ApoC-III (Rn00560743); ATP synthase H+ transporting mitochondrial F1 complex γ polypeptide 1 (Atp5c1 ) (Rn01487287_m1); ATP synthase H+ transporting mitochondrial F0 complex subunit F2 (Atp5j2 ) (Rn01409509_g1); γ-butyrobetaine hydroxylase (Bbox1 ) (Rn00575255_m1); cluster of differentiation 36 (Cd36 ) (Rn00580728_m1); Cpt1a (Rn00580702_m1); Cpt2 (Rn00563995_m1); carnitine O -acetyltransferase (Crat ) (Rn01758585_m1); somatic cytochrome c (Cycs ) (Rn00820639_g1); mitochondrial 2,4 dienoyl-CoA reductase 1 (Decr1 ) (Rn00589420_m1); diglyceride acyltransferase (Dgat )1 (Rn00584870_m1); Dgat2 (Rn01506787_m1); FA binding protein 1 (Fabp1 ) (Rn00664587_m1); Fasn (Rn00569117_m1); mitochondrial glycerol-3-phosphate dehydrogenase 2 (Gpd2 ) (Rn00562472_m1); hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) α subunit (Hadha ) (Rn00590828_m1); hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) β subunit (Hadhb ) (Rn00592435_m1); mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2 ) (Rn00597339_m1); Lpl (Rn00561482_m1); microsomal TG transfer protein (large subunit) (Mttp ) (Rn01522970_m1); NADH dehydrogenase (ubiquinone) 1α subcomplex 9 (Ndufa9 ) (Rn01462923_m1); Pparα (Rn00566193_m1); Pparδ (Rn00565707_m1); Pparγ (Rn00440945_m1); PPARγ coactivator 1α (Ppargc1a ) (Rn00580241_m1); protein kinase AMP-activated α1 catalytic subunit (Prkaa1 ) (Rn00569558_m1); protein kinase AMP-activated α2 catalytic subunit (Prkaa2 ) (Rn00576935_m1); Slc25a20 (Rn00588652_m1); solute carrier family 22 (organic cation/carnitine transporter) member 5 (Slc22a5 ) (Rn01471177_m1); mitochondrial transcription factor A (Tfam ) (Rn00580051_m1); N ε -trimethyllysine hydroxylase (Tmlhe ) (Rn00591314_m1); uncoupling protein (Ucp )2 (Rn01754856_m1); Ucp3 (Rn00565874_m1); VLDL receptor (Vldlr ) (Rn00565784_m1).

Techniques: Activity Assay, Pyrolysis Gas Chromatography

Journal: Scientific Reports

Article Title: A new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology

doi: 10.1038/srep17217

Figure Lengend Snippet: Biochemical markers of mitochondrial biogenesis in the reporter cells. ( A ) AMPK activation in HeLaNRF1/c4 cells. The cells were treated with 0.5 mM AICAR for 2, 5 or 10 min, with or without the AMPK inhibitor Compound C (10 μM). The levels of phosphorylated AMPK (pAMPK) and ACC (pACC) were analysed by western blotting. ( B ) The effect of AICAR (0.5 mM) on expression of NRF-1, TFAM and COX was measured at four different time points (1, 2, 3 and 6 days), by quantitative PCR. The data were calculated relative to the untreated control (Ctr) and are shown as mean ± S.D. of two experiments with triplicate measurements. ( C ) The amount of mtDNA relative to nuclear DNA (mtDNA copy number) was measured by quantitative PCR, in cells grown for 1, 2, 3 or 6 days in presence of AICAR (0.5 mM). The data were calculated relative to untreated control (Ctr), and are shown as mean ± S.D. of three experiments with triplicate measurements. ( D ) Kinetics of mitoGFP induction. The expression of mitoGFP was measured by flow cytometry at different time points during 6 days of treatment with AICAR (0.5 mM). The data represents mean ± S.D. of three experiments (10 000 cells per sample), calculated relative to untreated cells. ( E ) Dose-response of AICAR in HeLaNRF1/c4 cells. The cells were treated with increasing concentrations of AICAR for 6 days and mitoGFP was detected by flow cytometry (10 000 cells per sample). The data represent mean ± S.D. of n ≥ 3. *p

Article Snippet: Quantitative PCR was performed in the LightCycler 480 detection system (Roche, Basel, Switzerland) using the LightCycler 480 Probes master kit, and specific FAM probes and primers from Applied Biosystems: Nuclear respiratory factor 1; NRF-1 , Hs00602161_m1, Transcriptional Factor A mitochondrial; TFAM , Hs01082775_m1 and Cytochrome C Oxidase; Cox4i, Hs00971639_m1.

Techniques: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

Journal: Diabetes

Article Title: Inhibition of Class I Histone Deacetylases Unveils a Mitochondrial Signature and Enhances Oxidative Metabolism in Skeletal Muscle and Adipose Tissue

doi: 10.2337/db12-0548

Figure Lengend Snippet: HDAC3 ablation mimics the effect of class I HDAC inhibitors in a PGC-1α–dependent manner. A : Pgc-1α , Glut4 , Tfam , and Idh3α expression in C2C12 myotubes infected with adenoviruses expressing shRNA against Pgc-1α (■) or scramble control (□). Note that the effect of HDAC inhibitors is lost in the absence of Pgc-1α. B : Schematic representation of the HDACs known to be present on the Pgc-1α promoter (prom). C : Hdac3 protein levels in C2C12 myoblasts transfected with small interfering RNA against Hdac3 or control. D : Gene expression profile after silencing Hdac3 in C2C12 myoblasts. E and F : Hdac3 ChIP of C2C12 myotubes or skeletal muscle (SKM) of db/db mice treated with HDAC inhibitors. Bars represent presence of Hdac3 on the Pgc-1α promoter within the Mef2 or the cAMP-responsive element regions shown in B . A distal region was used as a negative control. Data are presented as means ± SD. * P

Article Snippet: Anti–acetyl-H3, anti-HDAC1, anti-cytochrome C (CytC), and anti–rabbit IgG (Cell Signaling); anti–acetyl-tubulin, anti–α-tubulin, anti–β-actin, anti-Tfam, and anti–mouse IgG (Sigma-Aldrich); anti-HDAC3 and anti–peroxisome proliferator–activated receptor (PPAR)γ (Santa Cruz); and anti-HDAC3 (ChIP), anti–uncoupling protein (UCP)1, and anti-LCAD (Abcam) antibodies were used.

Techniques: Pyrolysis Gas Chromatography, Expressing, Infection, shRNA, Transfection, Small Interfering RNA, Chromatin Immunoprecipitation, Mouse Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor ? Co-activator 1? (PGC-1?) and Sirtuin 1 (SIRT1) Reside in Mitochondria

doi: 10.1074/jbc.M109.070169

Figure Lengend Snippet: Assay of the presence of SIRT1 and PGC-1α in cross-linked mitochondrial nucleoids. A , scheme for purification of nucleoids ( N ) from mouse liver mitochondria. Fractions ( 1–14 ) were finally collected by pricking the bottom of the tube. B , presence of mtDNA in the collected fractions was assayed performing PCR analysis of D-loop region. The presence of genomic DNA was excluded by performing PCR analysis of actin gene. After cross-linking reversion, fractions were subjected to SDS-PAGE. Western blot analysis with mouse anti-cytochrome c oxidase subunit IV ( COX IV ) and mouse anti-cytochrome c ( Cyt c ) was carried out to determine the possible presence of protein contaminants. SIRT1, HSP60, TFAM, and SOD2 were detected by using specific rabbit antibodies. PGC-1α was detected by using both the Santa Cruz Biotechnology and the Calbiochem antibodies. C , cross-linked mitochondria from mouse liver were sonicated and immunoprecipitated with goat anti-TFAM or goat anti-PGC-1α antibody (Santa Cruz Biotechnology). DNA was extracted, and PCR analysis of the region +15,600/+15,868 of mtDNA was successively carried out. Input and immunoprecipitation with a control IgG were used as positive and negative controls. D , mitochondrial protein extracts from HEK293 cells were analyzed by oligonucleotide pulldown assay using TFAM biotinylated consensus oligonucleotide and Western blot analysis using Santa Cruz Biotechnology anti-TFAM, anti-SIRT1, and anti-PGC-1α antibody. Mito , total mitochondrial lysates; lanes 1 and 2 , two independent oligonucleotide pulldown experiments. Immunoblots reported are from one experiment representative of at least three that gave similar results.

Article Snippet: Samples were then pre-cleared with protein A/G-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C, and 500 μg/ml proteins were immunoprecipitated overnight at 4 °C using 4 μg of goat anti-PGC-1α or goat anti-TFAM (Santa Cruz Biotechnology) as positive control.

Techniques: Pyrolysis Gas Chromatography, Purification, Polymerase Chain Reaction, SDS Page, Western Blot, Sonication, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor ? Co-activator 1? (PGC-1?) and Sirtuin 1 (SIRT1) Reside in Mitochondria

doi: 10.1074/jbc.M109.070169

Figure Lengend Snippet: Determination of the association of SIRT1 and PGC-1α with mtDNA and TFAM by confocal microscopy in HeLa cells. A , mtDNA was labeled by BrdUrd incorporation followed by methanol-acetone fixing and immunostaining with mouse anti-BrdUrd. SIRT1 or PGC-1α was also immunostained using Santa Cruz Biotechnology antibodies. Cells were then visualized by confocal microscopy, and the level of superimposition of SIRT1 or PGC-1α-derived signals with that of BrdUrd was evaluated by calculation of the Pearson's correlation coefficient ( R ( r )). Values > 0.50 were considered to be significant. B , HeLa cells were fixed with paraformaldehyde and immunostained with goat anti-TFAM and rabbit Santa Cruz Biotechnology anti-PGC-1α or SIRT1. Cells were then visualized by confocal microscopy, and the level of superimposition of SIRT1 or PGC-1α-derived signals with that of TFAM was evaluated by calculation of the Pearson's correlation coefficient ( R ( r )). Values > 0.50 were considered to be significant. Scale bar, 10 μm.

Article Snippet: Samples were then pre-cleared with protein A/G-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C, and 500 μg/ml proteins were immunoprecipitated overnight at 4 °C using 4 μg of goat anti-PGC-1α or goat anti-TFAM (Santa Cruz Biotechnology) as positive control.

Techniques: Pyrolysis Gas Chromatography, Confocal Microscopy, Labeling, Immunostaining, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor ? Co-activator 1? (PGC-1?) and Sirtuin 1 (SIRT1) Reside in Mitochondria

doi: 10.1074/jbc.M109.070169

Figure Lengend Snippet: Detection of mitochondrial SIRT1 and PGC-1α in mitochondria purified from HeLa cells and mouse organs and in human platelets. A , scheme for purification of mitochondria from HeLa and mouse organs. B , 10 μg of proteins obtained from HeLa purified mitochondria ( Mito ) and nuclei-enriched pellets ( P1 ) were subjected to SDS-PAGE followed by Western blot analysis. The possible presence of nuclear protein contaminants was measured by incubating nitrocellulose membrane with rabbit anti-H2B. The effective mitochondria isolation was assessed by staining with rabbit anti-TFAM and mouse anti-cytochrome c oxidase sub IV ( COX IV ). PGC-1α and SIRT1 were detected by using rabbit Santa Cruz Biotechnology antibodies. C , 10 μg of proteins obtained from nuclei-enriched pellets ( P1 ) and mitochondria ( Mito ) isolated from mice brain ( B ), skeletal muscle ( M ), and liver ( L ) were subjected to SDS-PAGE followed by Western blot analysis. The possible presence of nuclear protein contaminants was measured by incubating nitrocellulose membrane with rabbit anti-lamin B. The effective mitochondrial isolation was assessed by staining with rabbit anti-SOD2. PGC-1α and SIRT1 were detected by using Santa Cruz Biotechnology antibodies. D , human platelets were purified from peripheral blood, and 20 μg of protein extracts were subjected to SDS-PAGE followed by Western blot analysis. The possible presence of nuclear proteins was measured by incubating the nitrocellulose membrane with rabbit anti-H2B. PGC-1α and SIRT1 were detected by using Santa Cruz Biotechnology antibodies. Immunoblots reported are from one experiment representative of at least three that gave similar results.

Article Snippet: Samples were then pre-cleared with protein A/G-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C, and 500 μg/ml proteins were immunoprecipitated overnight at 4 °C using 4 μg of goat anti-PGC-1α or goat anti-TFAM (Santa Cruz Biotechnology) as positive control.

Techniques: Pyrolysis Gas Chromatography, Purification, SDS Page, Western Blot, Isolation, Staining, Mouse Assay

Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor ? Co-activator 1? (PGC-1?) and Sirtuin 1 (SIRT1) Reside in Mitochondria

doi: 10.1074/jbc.M109.070169

Figure Lengend Snippet: Identification of mitochondrial multiprotein complexes by BN-PAGE in liver purified mitochondria. A , mitochondrial proteins were extracted from liver purified mitochondria ( mito ) using a lysis buffer maintaining native interaction in multiprotein complexes. Ten μg of proteins were loaded on blue native gel. The same samples were boiled in the presence of SDS and loaded in parallel with the respective native sample. Western blot ( WB ) analysis using rabbit Santa Cruz Biotechnology anti-SIRT1 or anti-PGC-1α was then performed. B , after BN-PAGE, a second SDS-PAGE dimension was carried out followed by Western blot analysis using rabbit Santa Cruz Biotechnology anti-PGC-1α or anti-SIRT1. MPC , multiprotein complexes. C , 10 μg of proteins were loaded on blue native gel, and Western blot using a rabbit anti-TFAM was carried out. 1 , multiprotein complex containing TFAM, PGC-1α, and SIRT1; 2 and 3, multiprotein complexes containing PGC-1α and SIRT1; 4 , multiprotein complex containing TFAM, and SIRT1; 5 multiprotein complex containing TFAM and PGC-1α; 6 , multiprotein complex containing TFAM-PGC-1α and TFAM-SIRT1; a–c , other TFAM multiprotein complexes. To visualize free TFAM protein, a major exposure was mandatory due to the lower cross-reactivity of TFAM antibody against native TFAM with respect to TFAM engaged in multiprotein complexes (see inset in TFAM immunoblot). Immunoblots reported are from one experiment representative of four that gave similar results.

Article Snippet: Samples were then pre-cleared with protein A/G-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C, and 500 μg/ml proteins were immunoprecipitated overnight at 4 °C using 4 μg of goat anti-PGC-1α or goat anti-TFAM (Santa Cruz Biotechnology) as positive control.

Techniques: Polyacrylamide Gel Electrophoresis, Purification, Lysis, Western Blot, Pyrolysis Gas Chromatography, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor ? Co-activator 1? (PGC-1?) and Sirtuin 1 (SIRT1) Reside in Mitochondria

doi: 10.1074/jbc.M109.070169

Figure Lengend Snippet: Assay of the presence of SIRT1 and PGC-1α in mitochondrial native nucleoids. Native nucleoids were obtained from isolated liver mitochondria as described under “Experimental Procedures.” After sucrose gradient, nucleoid-containing fractions were identified by detecting the presence of TFAM by dot blot (using goat anti-TFAM) and mtDNA by PCR analysis of the D-loop region. Western blot analyses with mouse anti-cytochrome c oxidase subunit IV ( COX IV ), mouse anti-cytochrome c ( Cyt c ), rabbit anti-SOD2, and rabbit anti-HSP60 were carried out to determine the possible presence of protein contaminants. Fractions 6–9 were considered to contain native nucleoids due to the absence of protein contaminants and to the presence of TFAM and mtDNA. PGC-1α and SIRT1 were detected by using Santa Cruz Biotechnology antibodies. Immunoblots reported are from one experiment representative of at least three that gave similar results.

Article Snippet: Samples were then pre-cleared with protein A/G-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C, and 500 μg/ml proteins were immunoprecipitated overnight at 4 °C using 4 μg of goat anti-PGC-1α or goat anti-TFAM (Santa Cruz Biotechnology) as positive control.

Techniques: Pyrolysis Gas Chromatography, Isolation, Dot Blot, Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: miR-504 mediated down-regulation of nuclear respiratory factor 1 leads to radio-resistance in nasopharyngeal carcinoma

doi:

Figure Lengend Snippet: miR-504 directly targets NRF1 and down-regulates the expression of the TFAM and OXPHOS complexes A. The interaction between miR-504 and NRF1 can be predicted in several databases shown by miRecords. B. Relative expression level of NRF1 mRNA in the CNE2 cell line at 48, 72, or 96 h after transfection of a miR-504 precursor (CNE2-miR-504) or its negative control (CNE2-NC). Data are shown as mean values ± S.D. of three experiments. There is no statistical significance among these groups. C. The protein expression level of NRF1 in the CNE2 cell line at 48, 72, or 96 h after transfection of a miR-504 precursor (CNE2-miR-504) or its negative control (CNE2-NC). D. The protein expression level of NRF1 in the CNE2-IR cell line at 48 h after transfection of an inhibitor of miR-504 (CNE2-IR-anti-miR-504) or its negative control (CNE2-IR-anti-NC). E. The putative miR-504 binding site and its mutant site in the 3′-UTR of NRF1 are shown (upper panel). The relative luciferase activities of cells expressing a pMIR-reporter plasmid, NRF1-3′-UTR plasmid, or NRF1-3′-UTR-MUT plasmid when co-transfected with a miR-504 precursor or its negative control are shown (lower panel). Data are shown as mean values ± S.D. of three experiments. The asterisks (**) indicate a significant difference (p

Article Snippet: Western blot analysis The antibodies for Western blot were as follows: NRF1 (sc-101102, Santa Cruz, CA, USA), TFAM (OAAB00276, Aviva System Biology, San Diego, CA, USA), MitoProfile® Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam) and β-actin (sc-8432, Santa Cruz).

Techniques: Expressing, Transfection, Negative Control, Binding Assay, Mutagenesis, Luciferase, Plasmid Preparation

Journal: International Journal of Endocrinology

Article Title: Functional Characterization of Preadipocytes Derived from Human Periaortic Adipose Tissue

doi: 10.1155/2017/2945012

Figure Lengend Snippet: PAT expressed proteins involved in thermogenesis. (a) Samples from SAT and PAT were obtained and immediately frozen in liquid nitrogen. mRNA was extracted and the detection of PGC-1 α and UCP-1 was performed by qPCR. (b) Precursor adipose cells (PAC) from SAT and PAT were induced to differentiate into mature adipocytes (MAT), and proteins were extracted to quantify the levels of PGC-1 α , UCP-1, CITED1, and TFAM by Western blotting. (c) Relative intensity of the protein bands was determined by densitometry. Data were normalized to the housekeeping protein GAPDH and expressed as means ± SD ( n = 4). ∗ p

Article Snippet: The membranes were blocked with 5% skimmed milk in 1x PBS containing 0.1% Tween 20 (PBS-T) and incubated with primary antibodies against proteins involved in thermogenesis or antibodies against adipokines as follows: rabbit anti-PGC-1α (1 : 1000, Abcam, Cambridge, MA, USA, cat. number ab54481), rabbit anti-TFAM (1 : 1000, Cell Signaling, Beverly, MA, USA, cat. number ab155117), rabbit anti-CITED1 (1 : 1000, Abcam, Cambridge, MA, USA, cat. number ab87978), rabbit anti-UCP-1 (1 : 1000, Abcam, cat. number ab155117), adiponectin (1 : 3000, Abcam, Cambridge, MA, USA, cat. number ab92501), FGF21 (1 : 2000, Abcam, Cambridge, MA, USA, cat. number ab171941), and FABP4 (1 : 3000, Abcam, Cambridge, MA, USA, cat. number ab92501).

Techniques: Pyrolysis Gas Chromatography, Real-time Polymerase Chain Reaction, Western Blot

Journal: Physiological Reports

Article Title: Acute and chronic effects of resistance training on skeletal muscle markers of mitochondrial remodeling in older adults, et al. Acute and chronic effects of resistance training on skeletal muscle markers of mitochondrial remodeling in older adults

doi: 10.14814/phy2.14526

Figure Lengend Snippet: RT acutely increased NRF1 but did not alter PGC‐1α or TFAM protein levels. Legend: Pre, protein levels at baseline; Acute, protein levels at 24 hr after the first training session; Chronic, protein levels at 72 hr after the last training session; *, significantly different from Pre ( p

Article Snippet: Primary antibodies were used to detect: Total OXPHOS Human Cocktail (Abcam Cat# ab110411, RRID:AB_2756818), COX IV (Cell Signaling Technology Cat# 4850, RRID:AB_2085424), PGC‐1α (GeneTex Cat# GTX37356, RRID:AB_11175466), NRF1 (GeneTex Cat# GTX103179, RRID:AB_11168915), TFAM (Abnova Corporation Cat# H00007019‐D01P, RRID:AB_1715621), Mfn1 (Cell Signaling Technology Cat# 14739, RRID:AB_2744531), Mfn2 (BioVision Cat# 3882‐100, RRID:AB_2142625), Opa1 (Cell Signaling Technology Cat# 67589, RRID:AB_2799728), Fis1 (Abcam Cat# ab71498, RRID:AB_1271360), Drp1 (Novus Cat# NB110‐55288SS, RRID:AB_921147), Pink1 (Cell Signaling Technology Cat# 6946, RRID:AB_11179069), and Parkin (Cell Signaling Technology Cat# 2132, RRID:AB_10693040).

Techniques: Pyrolysis Gas Chromatography