Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) CRISPR-Cas9 mediated stable targeting of ACO2 gene in C4-2 cells using three independent sgRNAs were confirmed by immunoblotting Wild Type (WT) and Knockdown (ACO2-KD) pooled clones. Actin was used as a loading control.

Article Snippet: Cells were transfected either with 5 μg WT or K258R mutated ACO2 plasmids (Origene, RC204307) for 48 hours.

Techniques: CRISPR, Western Blot, Clone Assay

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) C4-2-WT and C4-2-ACO2-KD cells (n=5, each group) were used to measure oxygen consumption rates (OCR) sequentially before and after the addition of oligomycin (2 μM), FCCP (1 μM), and antimycin A (1 μM) by XF24 Extracellular Flux Analyzer. Data are shown in mean ± S.D. Each time point is statistically significant. **** P<0.000001, two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Cells were transfected either with 5 μg WT or K258R mutated ACO2 plasmids (Origene, RC204307) for 48 hours.

Techniques:

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) V5-tagged-ACO2 adenovirus were transduced to C4-2 cells fractionated into cytosolic (Cyto), mitochondrial (Mito), and nuclear (Nuc) compartments followed by immunoblotting with cytosolic, mitochondrial, and nuclear proteins β-tubulin, Tom20 and lamin A/C respectively.

Article Snippet: Cells were transfected either with 5 μg WT or K258R mutated ACO2 plasmids (Origene, RC204307) for 48 hours.

Techniques: Western Blot

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) PC3 cells were transfected with myc-ACO2-WT or myc-ACO2-K258R for 48 hours (n=4, each group). Immunofluorescence staining was performed with anti-myc (green) while being counterstained with mitotracker (red) and DAPI (blue) followed by confocal microscopy. Scale bars, 10 μm.

Article Snippet: Cells were transfected either with 5 μg WT or K258R mutated ACO2 plasmids (Origene, RC204307) for 48 hours.

Techniques: Transfection, Immunofluorescence, Staining, Confocal Microscopy

Journal: Cancer research

Article Title: Transcriptional repression of SIRT3 potentiates mitochondrial aconitase activation to drive aggressive prostate cancer to the bone

doi: 10.1158/0008-5472.CAN-20-1708

Figure Lengend Snippet: (A) HEK293T cells were transduced with WT-SIRT3-Flag or HA-SIRT3-H248Y plasmid and immunoblotted for ACO2, HA, Flag, Actin, and Ac-K in input depicting WT-SIRT3-Flag or HA-SIRT3-H248Y expression. Transfected cell lysates were used for endogenous ACO2 and IgG (control) immunoprecipitation, followed by immunoblotting for ACO2 and Ac-K.

Article Snippet: Cells were transfected either with 5 μg WT or K258R mutated ACO2 plasmids (Origene, RC204307) for 48 hours.

Techniques: Transduction, Plasmid Preparation, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: Journal of Ovarian Research

Article Title: Bushen Huatan formula alleviates polycystic ovary syndrome in rats by activating the PI3K/Akt pathway to inhibit GSDMD-mediated pyroptosis and mitochondrial damage

doi: 10.1186/s13048-025-01887-w

Figure Lengend Snippet: The effect of BSHT on ovarian pyroptosis and mitochondrial damage. A Western blot analysis and quantification of protein expression of GSDMD-N, C-caspase-1, and IL-1β; B Western blot analysis and quantification of protein expression of ACO2, GSDMD-N, and Cytochrome C. (*p < 0.05, **p < 0.01, ***p < 0.001 BSHT group vs. Model group, Metformin group vs. Model group, #p < 0.05 ##p < 0.01, ###p < 0.001 Model group vs. Control group.) (n=6)

Article Snippet: Primary antibodies included p-PI3K, PI3K, p-AKT, and AKT for PI3K/AKT signaling pathway proteins; GSDMD-N (Cat No. 20770-1-AP, Proteintech, China), C-caspase-1, and IL-1β for pyroptosis-related proteins; Beclin-1 (Cat No. 11306-1-AP, Proteintech, China), ACO2, and Cytochrome C for autophagy and mitochondrial markers; with β-Actin used as the loading control.

Techniques: Western Blot, Expressing, 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: 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, Control, Staining, Live Cell Imaging, Membrane, Comparison, 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