Journal: Stem Cells International

Article Title: Energy Metabolism and Lipidome Are Highly Regulated during Osteogenic Differentiation of Dental Follicle Cells

doi: 10.1155/2022/3674931

Figure Lengend Snippet: Expression of glycolysis and citric acid cycle markers during osteogenic differentiation of DFCs. (a) DFCs were cultured in osteogenic differentiation medium (ODM), BMP2 differentiation medium, or control medium (DMEM) for one, seven, 14, and 28 days before protein expression of the glycolysis markers hexokinase I, hexokinase II, phosphofructokinase, glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH), pyruvate kinase M1/2, pyruvate kinase M2, and lactate dehydrogenase A, and protein expression of the citric acid cycle markers aconitase 2, isocitrate dehydrogenase 1, isocitrate dehydrogenase 2, dihydrolipoamide succinyltransferase (DLST), fumarase, citrase synthase, mitochondrial pyruvate carrier 1, and mitochondrial pyruvate carrier 2 were determined by western blot analysis. Quantification results and statistical analysis are shown in the Supplementary Table . (b–d) DFCs were cultured in osteogenic differentiation medium (ODM), BMP2 differentiation medium, or control medium (DMEM) for seven days before the amount of produced L-lactate (b) and hexokinase activity (c) were determined as markers of glycolysis and malate dehydrogenase activity was measured (d) as marker of citric acid cycle. Results are shown as means + standard deviation, and Student's t -test was performed to compare differentiation medium with control medium. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C in a primary antibody against hypoxia-inducible factor 1-alpha (HIF-1 α ), cytochrome c, prohibitin 1, cytochrome c oxidase subunit 4 (COX IV), pyruvate dehydrogenase, succinate dehydrogenase complex subunit A (SDHA), heat shock protein 60 (HSP60), voltage-dependent anion channel (VDAC), hexokinase I, hexokinase II, phosphofructokinase, glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH), pyruvate kinase M1/2, pyruvate kinase M2, lactate dehydrogenase A, aconitase 2, isocitrate dehydrogenase 1, isocitrate dehydrogenase 2, dihydrolipoamide succinyltransferase (DLST), fumarase, citrase synthase, mitochondrial pyruvate carrier 1, mitochondrial pyruvate carrier 2, acetyl-CoA carboxylase, phospho-acetyl-CoA carboxylase (Ser79), ATP-citrate lyase, phospho-ATP-citrate lyase (Ser455), acetyl-CoA synthetase, acyl-CoA synthetase, fatty acid synthase (all Cell Signaling Technology, Danvers, MA, USA), or elongation of very long chain fatty acids protein 6 (ELOVL6) (Abcam, Cambridge, UK).

Techniques: Expressing, Cell Culture, Western Blot, Produced, Activity Assay, Marker, Standard Deviation

Journal: Journal of Cancer Prevention

Article Title: Breast Cancer Selective Disruption of Actin Cytoskeleton by Diallyl Trisulfide

doi: 10.15430/JCP.2022.27.2.101

Figure Lengend Snippet: (A) Reactome pathway enrichment analysis of downregulated genes by DATS treatment in MCF-10A and SK-BR-3 cell lines. (B) Immunoblot analysis for ACO2 and DLST in MCF-10A and SK-BR-3 cells treated with DMSO (control) or indicated doses of DATS for specified time points. The numbers above bands represent fold change in expression relative to corresponding DMSO-treated controls. (C) Quantification of ACO2 and DLST expression in SK-BR-3 cells. Results combined from three independent experiments are shown as mean ± SD (n = 3) and statistical analysis was done by one-way ANOVA followed by Dunnett’s test. DATS, diallyl trisulfide; DMSO, dimethyl sulfoxide, DLST, dihydrolipoamide S-succinyltransferase; ACO2, aconitase 2; HDR, Homology directed repair; ATR, ataxia telangiectasia and Rad3 related; AP, abasic sites; PCNA, proliferating cell nuclear antigen.

Article Snippet: Anti-aconitase 2 (ACO2) antibody and anti-dihydrolipoamide S-succinyltransferase (DLST) antibody were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing

Journal: Cell reports

Article Title: Aging-dependent mitochondrial dysfunction mediated by ceramide signaling inhibits antitumor T cell response

doi: 10.1016/j.celrep.2021.109076

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Aco2 , Cell Signaling , Cat# 6922S, RRID:AB_10828218.

Techniques: Transduction, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling, Lysis, CyQUANT Assay, Proliferation Assay, Activity Assay, In Vitro, ATP Bioluminescent Assay, SYBR Green Assay, RNA Sequencing Assay, Expressing, Generated, Software

Journal: Cell reports

Article Title: Aging-dependent mitochondrial dysfunction mediated by ceramide signaling inhibits antitumor T cell response

doi: 10.1016/j.celrep.2021.109076

Figure Lengend Snippet: (A and B) Viability of naive (A) or TCR activated (B) T cells isolated from Y or A mice was measured by trypan blue exclusion assay. (C) Flow cytometry analyses show IFN-γ suppression in activated T cells isolated from A compared to Y mice. Data are means ± SDs from 3 independent experiments (n = 3). *p < 0.05 as determined by Student’s t test. (D) The ability of aging Pmel T cells to kill B16 melanoma cancer cells in co-cultures is impaired compared to T cells isolated from Y mice. Data are means ± SDs from 3 independent experiments (n = 3). **p < 0.01 as determined by Student’s t test. (E and F) Mitophagy of Y (isolated from 2- and 4-month-old mice) and A (isolated from 8- and 18-month-old mice) activated T cells were measured by live-cell imaging using confocal microscopy stained for mitochondria (MITO; MitoTracker Red [MTR]) and lysosomes (LysoTracker Green [LTG]). Scale bars, 1 μm. Micrographs represent at least 3 independent experiments of Y and A T cells (E). The right panel shows the quantification of colocalization extracted from the coefficient of colocalization (Rc). White arrows indicate merged (yellow). Scale bars, 1 μm. Quantification of colocalization extracted from the coefficient of colocalization (Rc) using the Fiji software (F). Data are means ± SDs from 3 independent experiments (n = 3). *p < 0.05 as determined by Student’s t test. (G) Live-cell imaging of naive Y and A T cells, stained MITO (MTR) and lysosomes (LTG). Scale bars, 1 μm. Micrographs represent at least 3 independent experiments of Y and A T cells. The bottom panel shows the quantification of colocalization extracted from the coefficient of colocalization (Rc). Data are means ± SDs from 3 independent experiments (n = 3). (H and I) Western blot measuring the levels of ACO2 and P-S637-Drp1 in activated T cells from Y (2- and 4-month-old), and A (8- and 18-month-old) mice. Protein expression was normalized to β-actin. (I) Western blot measuring cytoplasmic levels of LC3B-I, LC3B-II, and ACO2 (H), or total-Drp1, P-(S637)-Drp1, and P-(S616)-Drp1 (I), in activated T cells from Y and A mice. (J and K) Western blot to detect P-S637-Drp1 (J) and ACO2 (K) in activated T cells with/without mitophagy inhibitor mDivi (Md). Data are means ± SDs from 3 independent experiments (n = 3). *p < 0.05, **p < 0.01 as determined by Student’s t test.

Article Snippet: Anti-Aco2 , Cell Signaling , Cat# 6922S, RRID:AB_10828218.

Techniques: Isolation, Trypan Blue Exclusion Assay, Flow Cytometry, Live Cell Imaging, Confocal Microscopy, Staining, Software, Western Blot, Expressing

Journal: Cell reports

Article Title: Aging-dependent mitochondrial dysfunction mediated by ceramide signaling inhibits antitumor T cell response

doi: 10.1016/j.celrep.2021.109076

Figure Lengend Snippet: (A) Confocal microscopy for Y and A T cells with/without FB1 dual labeled with TOM20 (red, mitochondrial marker), and ceramide (green) fluorescent antibodies. White arrows indicate merged (yellow). Scale bars, 1 μm. Images represent at least 3 independent experiments. The right panel shows the quantification of colocalization extracted from the coefficient of colocalization (Rc). Data are means ± SDs from 3 independent experiments (n = 3). (B) Autophagy was measured staining with CytoID followed by flow cytometry of 3-day activated Y and A T cells with/without FB1. Data show means ± SDs from 3 independent experiments (n = 3). **p < 0.01 as determined by Student’s t test. (C) High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) measurements of C14 ceramide (left), C16 ceramide (center), and C18 ceramide (right) in activated T cells isolated from Y and A mice. Data are means ± SDs from 3 experiments (n = 3). *p < 0.05 as determined by Student’s t test. (D) Mitophagy was detected by live-cell imaging for measuring the colocalization of MTR-LTG in T cells obtained from Y and A WT, CerS5 −/− and CerS6 −/− mice. Quantification of colocalization was performed using the coefficient of colocalization (Rc). Data are means ± SDs from at least 3 experiments (n = 3). ***p < 0.001 as determined by Student’s t test. (E–G) LC3/autophagy activation was measured by cyto-ID (E), and protein abundance of P-S637-Drp1 and LCBI/LCBII (F) or ACO2 and actin (G) were measured using extracts of activated T cells isolated from Y and A WT, CerS5 −/− , and/or CerS6 −/− mice. These data represent at least 3 independent experiments (n = 3). Data are means ± SDs from 3 independent experiments (n = 3).

Article Snippet: Anti-Aco2 , Cell Signaling , Cat# 6922S, RRID:AB_10828218.

Techniques: Confocal Microscopy, Labeling, Marker, Staining, Flow Cytometry, High Performance Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Isolation, Live Cell Imaging, Activation Assay

Journal: Cell reports

Article Title: Aging-dependent mitochondrial dysfunction mediated by ceramide signaling inhibits antitumor T cell response

doi: 10.1016/j.celrep.2021.109076

Figure Lengend Snippet: (A) Mitochondrial accumulation of C14 ceramide was measured using mass spectrometry/lipidomics in activated T cells obtained from Y and A SphK2 −/− (SK2) mice in MAMs and MITO-enriched fractions. (B) Colocalization of ceramide (green) and TOM20 (red) was measured to detect mitophagy in activated T cells isolated from Y and A WT and SphK2 −/− (SK2) mice. Lower panel indicates the quantification of colocalization (Rc). Data are means ± SDs from 3 independent experiments (n = 3). **p < 0.01 as determined by Student’s t test. (C) P(S637)-Drp1 and ACO2 protein abundance was measured by western blotting in T cells isolated from Y and A WT or A SphK2 −/− (SK2) and SphK1 −/− (SK1) mice. (D and E) TCR-activated T cells obtained from Y and A WT or SphK2 −/− (SK2) mice were used for the detection of LC3/autophagy by cyto-ID (D) and ACO2 degradation by mitophagy using western blotting (E). Actin was used as a loading control (E). These studies represent 3 independent experiments (n = 3). **p < 0.01 as determined by Student’s t test in (D).

Article Snippet: Anti-Aco2 , Cell Signaling , Cat# 6922S, RRID:AB_10828218.

Techniques: Mass Spectrometry, Isolation, Western Blot

Journal: Cell reports

Article Title: Aging-dependent mitochondrial dysfunction mediated by ceramide signaling inhibits antitumor T cell response

doi: 10.1016/j.celrep.2021.109076

Figure Lengend Snippet: (A) T cells obtained from Y and A WT or 1 or 2 alleles targeted mutation of murine PKA (Prkaa2) PKA +/fl CD4 cre and PKA fl/fl CD4 cre mice were analyzed using live-cell microscopy for MTR-LTG colocalization. Arrows indicate merged (yellow); scale bars: 1 μm. Images represent at least 3 independent experiments. The right panel shows the quantification of colocalization extracted from the coefficient of colocalization (Rc). Data are means ± SDs from 3 independent experiments (n = 3). **p < 0.01 as determined by Student’s t test. (B and C) Western blotting was performed to detect PKAc (cat) (B) or P-(S637)-Drp1 (C) in T cells obtained from Y and A WT or Y Prkaa2 knockout mice (PKA +/+ and PKA +/fl CD4 cre and PKA fl/fl CD4 cre ). The quantification of PKAc abundance was shown in the right panel in (B). Data are means ± SDs from 3 independent experiments (n = 3). (D–F) Mitophagy/autophagy was detected in T cells isolated from Y and A PKA +/+ , and PKA +/fl CD4 cre , and PKA fl/fl CD4 cre mice in the absence/presence of mDivi for the measurement of cyto-ID (D) using flow cytometry, or P(S637)-Drp1 and ACO2 using western blotting. Data are means ± SDs from at least 3 experiments (n = 3). **p < 0.01 as determined by Student’s t test.

Article Snippet: Anti-Aco2 , Cell Signaling , Cat# 6922S, RRID:AB_10828218.

Techniques: Mutagenesis, Microscopy, Western Blot, Knock-Out, Isolation, Flow Cytometry

Journal: Cell reports

Article Title: Aging-dependent mitochondrial dysfunction mediated by ceramide signaling inhibits antitumor T cell response

doi: 10.1016/j.celrep.2021.109076

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Aco2 , Cell Signaling , Cat# 6922S, RRID:AB_10828218.

Techniques: Transduction, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling, Lysis, CyQUANT Assay, Proliferation Assay, Activity Assay, In Vitro, ATP Bioluminescent Assay, SYBR Green Assay, RNA Sequencing Assay, Expressing, Generated, Software

Journal: Scientific Reports

Article Title: Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy

doi: 10.1038/s41598-020-73557-4

Figure Lengend Snippet: Identification of the deletion c.1699_1749del51 in the ACO2 gene in a patient with dominant inherited optic nerve atrophy. ( a ) Family pedigree of the patient. Grey symbol: family members carrying the ACO2 deletion c.1699-1749del51. Red point: clinically affected family member with optic atrophy. Arrow head points to the index patient, from whom we obtained skin fibroblasts for the present study. Deceased individuals are marked with a line. ( b ) Results of Sanger sequencing of genomic DNA obtained from a healthy individual ( ACO2 WT) and the index patient ( ACO2 mutant), confirming the identified deletion c.1699-1749del51. ( c ) Visual field from the ophthalmologic examination of the patient (done in 2011) showing central scotoma of the left (LE) and the right eyes (RE). Dots represent defects surrounded by normal visual field, called scotomas (range: white > 5 dioptries (dB) to black > 30 dB). ( d ) Funduscopy of the ophthalmologic examination (done in 2011). Temporal paleness of the optic nerve in both eyes. ( e ) Retinal fibre layer of the optic nerve heads show thinning of the optical nerve fibers in the temporal regions via spectral domain optical coherence tomography (SD-OCT). The retinal fiber layer thickness is shown on the upper right side along the circular scan line (highlighted by green color on the upper left panel). The normal layer thickness is indicated by the green area on the lower right panel. The pathologically thinning of the layer in the temporal sectors of both eyes is indicated by yellow and red color. N nasal, NS/NI nasal superior/inferior, T temporal, TS/TI temporal superior/inferior. ( f ) Overall structure of the human ACO2 protein. The substrate-binding region is highlighted in blue. The region affected by the deletion is highlighted in red. ( g ) The stabilizing interactions between the segment 571–583 affected by the deletion (red backbone) and segment 656–684 (green), which passes through the active site and interacts with the substrate. Salt bridges are shown as black dashed lines, and the combined π-π and cation-π interactions are shown as blue dashed line.

Article Snippet: Proteins were blotted on nitrocellulose membrane (Invitrogen GmbH, Karlsruhe, Germany) by using the iBlot 2 device (Invitrogen GmbH, Karlsruhe, Germany) for 7 min at 20 V. Proteins of interest were labeled with primary antibodies against ACO2 (anti-rabbit; Abcam: ab129069; dilution: 1:1000), β-Actin (anti-mouse; Cell signal: 37005; dilution: 1:5000), TOM20 (anti-rabbit; Santa Cruz: Sc-11415; dilution: 1:1000) and secondary antibodies goat anti-mouse IgG (Novex: A24524; dilution: 1:10,000) or goat anti-rabbit IgG (Novex: A24537; 1:5000), respectively.

Techniques: Sequencing, Mutagenesis, Tomography, Binding Assay

Journal: Scientific Reports

Article Title: Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy

doi: 10.1038/s41598-020-73557-4

Figure Lengend Snippet: The deletion c.1699_1749del51 in the ACO2 gene leads to a growth defect of a Δaco1 yeast strain. ( a ) Drop dilution assay of yeast grown on galactose medium or ( b ) on ethanol medium at 30 °C or 35 °C and at different dilutions (optic densities (OD) ranging from 0.1, 0.01, 0.001 to 0.0001). Yeast cells with deletion of the aco1 gene expressed either human ACO2 wild-type (WT) or the ACO2 deletion-mutant (ACO2.mut) or ACO2-S112R, respectively. Expression of an empty Yep51 vector served as negative control.

Article Snippet: Proteins were blotted on nitrocellulose membrane (Invitrogen GmbH, Karlsruhe, Germany) by using the iBlot 2 device (Invitrogen GmbH, Karlsruhe, Germany) for 7 min at 20 V. Proteins of interest were labeled with primary antibodies against ACO2 (anti-rabbit; Abcam: ab129069; dilution: 1:1000), β-Actin (anti-mouse; Cell signal: 37005; dilution: 1:5000), TOM20 (anti-rabbit; Santa Cruz: Sc-11415; dilution: 1:1000) and secondary antibodies goat anti-mouse IgG (Novex: A24524; dilution: 1:10,000) or goat anti-rabbit IgG (Novex: A24537; 1:5000), respectively.

Techniques: Dilution Assay, Mutagenesis, Expressing, Plasmid Preparation, Negative Control

Journal: Scientific Reports

Article Title: Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy

doi: 10.1038/s41598-020-73557-4

Figure Lengend Snippet: Disease-associated ACO2 mutations cause impaired mtDNA maintenance. ( a ) Representative image of Western blot analysis of ACO2 protein. ( b ) Quantification of Western blot analysis of ACO2 protein levels normalized to β-Actin. Significance calculated by Mann–Whitney test (n = 3). ( c ) Western blot image and ( d ) the corresponding quantification of Tom20 protein, normalized to β-Actin. Significance calculated by Mann–Whitney test (n = 3). ( e ) Biochemical measurement of ACO2 enzyme activity in mitochondrial fractions from fibroblasts. Significance calculated by Mann–Whitney test (n = 5). ( f ) mtDNA copy number was analyzed by RT-PCR and indicated as ratio of the copy numbers of the mitochondrial gene ND1 to the nuclear encoded gene B2M. Significance was calculated by Mann–Whitney test (n = 9). ( g ) mtDNA transcription was analyzed by RT-PCR and indicated as ratio of the copy numbers of the D-Loop to the mitochondrial gene ND1. Significance was calculated by Mann–Whitney test (n = 3). ( h ) Major arc deletions in the mitochondrial genome were analyzed by RT-PCR and indicated as the ratio of the copy numbers of ND4, which is located on the minor arc, to ND1, which is located on the major arc of the mtDNA. Significance was calculated by Mann–Whitney test (n = 3). All data were indicated as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: Proteins were blotted on nitrocellulose membrane (Invitrogen GmbH, Karlsruhe, Germany) by using the iBlot 2 device (Invitrogen GmbH, Karlsruhe, Germany) for 7 min at 20 V. Proteins of interest were labeled with primary antibodies against ACO2 (anti-rabbit; Abcam: ab129069; dilution: 1:1000), β-Actin (anti-mouse; Cell signal: 37005; dilution: 1:5000), TOM20 (anti-rabbit; Santa Cruz: Sc-11415; dilution: 1:1000) and secondary antibodies goat anti-mouse IgG (Novex: A24524; dilution: 1:10,000) or goat anti-rabbit IgG (Novex: A24537; 1:5000), respectively.

Techniques: Western Blot, MANN-WHITNEY, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy

doi: 10.1038/s41598-020-73557-4

Figure Lengend Snippet: ACO2-mutant fibroblasts display reduced mitochondrial respiratory function. ( a ) Overview of measurement of oxygen consumption rate (OCR) in whole fibroblasts sequentially treated with 1 µM Oligomycin, 250 nM FCCP and 5 µM Antimycin A + Rotenone. OCR data were normalized to the total protein concentration in each well after cell lysis. Data indicated as mean ± SEM, (n = 5). ( b ) Basal respiration, ( c ) maximal respiration, ( d ) spare respiratory capacity, ( e ) proton leak, ( f ) ATP production and ( g ) coupling efficiency calculated from OCR data shown in ( a ). Data indicated as mean ± SEM. Significance calculated by Mann–Whitney test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, (n = 5). ( h ) Overview of measurement of the extra-cellular acidification rate (ECAR) in whole fibroblasts. During measurement, cells were sequentially treated with 1 mM Glucose, 10 µM Oligomycin and 10 mM 2-deoxyglucose (2-DG). ECAR data were normalized to the total protein concentration in each well after cell lysis. Data indicated as mean ± SEM, (n = 3). ( i ) Glycolysis rate, ( j ) Glycolytic capacity, ( k ) Glycolytic reserve and ( l ) non-glycolytic acidification were calculated from ECAR data shown in ( h ). Data indicated as mean ± SEM. * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Mann–Whitney test (n = 3).

Article Snippet: Proteins were blotted on nitrocellulose membrane (Invitrogen GmbH, Karlsruhe, Germany) by using the iBlot 2 device (Invitrogen GmbH, Karlsruhe, Germany) for 7 min at 20 V. Proteins of interest were labeled with primary antibodies against ACO2 (anti-rabbit; Abcam: ab129069; dilution: 1:1000), β-Actin (anti-mouse; Cell signal: 37005; dilution: 1:5000), TOM20 (anti-rabbit; Santa Cruz: Sc-11415; dilution: 1:1000) and secondary antibodies goat anti-mouse IgG (Novex: A24524; dilution: 1:10,000) or goat anti-rabbit IgG (Novex: A24537; 1:5000), respectively.

Techniques: Mutagenesis, Protein Concentration, Lysis, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy

doi: 10.1038/s41598-020-73557-4

Figure Lengend Snippet: ACO2-mutant fibroblasts are more susceptible to oxidative stress. ( a ) Representative images of control and ACO2-mutant fibroblasts stained with MitoTracker green FM for live cell imaging of mitochondria. Images were obtained with a 40 × objective. Scale bars indicate 50 µm. Images were used to analyse parameters of mitochondrial morphology, indicating ( b ) mitochondrial length, reflected by aspect ratio and ( c ) mitochondrial branching, reflected by form factor. Data indicated as mean ± SEM (n = 4). ( d ) Mitochondrial membrane potential of fibroblasts was analyzed by FACS. Cells were treated with 5 nM Valinomycin for 14 h in order to decrease the mitochondrial membrane potential and afterwards stained with TMRE. Data indicated as mean ± SEM, significance calculated by 2way ANOVA with post hoc Tukey’s multiple comparison test (n = 4). ( e ) Mitochondrial superoxide production was measured in fibroblasts using FACS analysis of MitoSOX staining. Cells were first treated with 20 nM Piericidin A for 14 h in order to inhibit the activity of the respiratory chain complex I. Data indicated as mean ± SEM, significance calculated by Mann–Whitney test (n = 3). ( f ) Cell viability was assessed using the Lactic Acid Dehydrogenase (LDH) assay. Fibroblasts were incubated with 5 mM H 2 O 2 for 4 h and afterwards the proportion of cell death was calculated from the amount of released LDH (n = 5). Data indicated as mean ± min./max. values. Significance calculated by Mann–Whitney test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: Proteins were blotted on nitrocellulose membrane (Invitrogen GmbH, Karlsruhe, Germany) by using the iBlot 2 device (Invitrogen GmbH, Karlsruhe, Germany) for 7 min at 20 V. Proteins of interest were labeled with primary antibodies against ACO2 (anti-rabbit; Abcam: ab129069; dilution: 1:1000), β-Actin (anti-mouse; Cell signal: 37005; dilution: 1:5000), TOM20 (anti-rabbit; Santa Cruz: Sc-11415; dilution: 1:1000) and secondary antibodies goat anti-mouse IgG (Novex: A24524; dilution: 1:10,000) or goat anti-rabbit IgG (Novex: A24537; 1:5000), respectively.

Techniques: Mutagenesis, Staining, Live Cell Imaging, Activity Assay, MANN-WHITNEY, Lactate Dehydrogenase Assay, Incubation

Journal: Scientific Reports

Article Title: Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy

doi: 10.1038/s41598-020-73557-4

Figure Lengend Snippet: ACO2 enzyme activity correlates with the severity of the clinical phenotype, but not with the protein amount of ACO2. Overview of patients with mutations in ACO2 from different studies, showing the correlation between ACO2 enzyme activity, protein level and severity of the clinical phenotype. Patients with mild clinical phenotype are represented with green dots, patients with intermediate phenotype are highlighted by orange dots and patients depicted by red dots show severe clinical phenotypes.

Article Snippet: Proteins were blotted on nitrocellulose membrane (Invitrogen GmbH, Karlsruhe, Germany) by using the iBlot 2 device (Invitrogen GmbH, Karlsruhe, Germany) for 7 min at 20 V. Proteins of interest were labeled with primary antibodies against ACO2 (anti-rabbit; Abcam: ab129069; dilution: 1:1000), β-Actin (anti-mouse; Cell signal: 37005; dilution: 1:5000), TOM20 (anti-rabbit; Santa Cruz: Sc-11415; dilution: 1:1000) and secondary antibodies goat anti-mouse IgG (Novex: A24524; dilution: 1:10,000) or goat anti-rabbit IgG (Novex: A24537; 1:5000), respectively.

Techniques: Activity Assay

Journal: Cell reports

Article Title: Autophagy Ablation in Adipocytes Induces Insulin Resistance and Reveals Roles for Lipid Peroxide and Nrf2 Signaling in Adipose-Liver Crosstalk

doi: 10.1016/j.celrep.2018.10.040

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-Aco2 , , Cell Signaling Technology , , Cat#6922S; RRID: AB_10828218.

Techniques: Enzyme-linked Immunosorbent Assay, TBARS Assay, Activity Assay, Recombinant