Journal: Frontiers in Cellular Neuroscience

Article Title: CoA synthase plays a critical role in neurodevelopment and neurodegeneration

doi: 10.3389/fncel.2024.1458475

Figure Lengend Snippet: Molecular and biochemical characterization of GFAP-Coasy mice. (A) Relative Coasy mRNA expression at 15 days and 6 months in Ctrl (n = 4) and GFAP-Coasy (n = 4) mice brain, tested by qPCR. (B) Representative images of Western blot analysis and (C–J) densitometric quantification of (C) Coasy, (D) Dcx, (E) Gfap, (F) Ft-H, (G) Ft-L, (H) Dmt1, (I) TfR1, and (J) Aco2 at 15 days and 6 months in Ctrl and GFAP-Coasy mice brain. (K) Specific activity of mitochondrial respiratory chain complex I, expressed as nmoles/min/mg of protein. * p < 0.05, ** p < 0.01; *** p < 0.001 (two-way ANOVA).

Article Snippet: A quantity of 10 to 35 μg of total proteins were separated using SDS-PAGE and then electroblotted onto a nitrocellulose membrane, which was subsequently incubated with specific antibodies for COASY (PA5-28696, Thermofisher Scientific, Waltham, MA, United States), DCX (#4604, Cell Signaling, Danvers, MA, United States), GFAP (MAB360, Millipore, Burlington, MA, United States), Ft-L and Ft-H , GAPDH (ab181602, Abcam, Cambridge, United Kingdom), TfR1 (13–6,800, Thermofisher Scientific, Waltham, MA, United States), DMT1 (sc-166884, Santa Cruz Biotechnology, Dallas, TX, United States), ACO2 (NBP1-32781, Novus Biologicals, Centennial, CO, United States), NCOA4 (sc-373739, Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Expressing, Western Blot, Activity 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) 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: Molecular Therapy. Methods & Clinical Development

Article Title: Exogenous expression of ATP8, a mitochondrial encoded protein, from the nucleus in vivo

doi: 10.1016/j.omtm.2024.101372

Figure Lengend Snippet: Transgenic optimized ATP8 localizes to mitochondria and incorporates into ATP synthase complex Cytoplasmic (Cyto.) and mitochondrial (Mito.) fractions were isolated from liver from 50-week-old transgenic C57BL/6J(mtC57BL/6J) and C57BL/6J(mtFVB) mice. Denaturing PAGE western blots were used to assess the compartmentalization of oATP8 to the mitochondria by probing for oATP8 using mouse anti-FLAG, cytosolic contents using rabbit anti-PGK1, and mitochondrial protein using mouse anti-ACO2. GAPDH was used as a loading control. The animal ID alongside the gender (“M” for male and “F” for female) is listed at the top of the lanes. Cytoplasmic fractions were run with ∼50 μg protein and mitochondrial fractions with ∼35 μg protein per lane ( n = 4 biological replicates; 2 males and 2 females) (A). The immunoblot bands of oATP8 were quantified by densitometry analysis (using ImageJ), normalized to aconitase for mitochondrial fractions, and to PGK1 for cytoplasmic fractions (B). Values are expressed as means ± standard error of the mean. Blue Native PAGE western blots using 25 μg protein from purified liver mitochondrial fractions of 12-week-old non-transgenic and transgenic C57BL/6J(mtC57BL/6J) and C57BL/6J (mtFVB mice). Integration of oATP8 into ATP synthase complex was detected by ATP synthase complex monomer (∗) and dimer (∗∗) formation using mouse anti-FLAG antibody (Ci). ATP5O/OSCP (Cii), and ATP6 (Ciii) were probed as controls for ATP synthase complex proteins ( n = 3 animals).

Article Snippet: The following antibodies were used in this study: anti-FLAG (cat. no. F1804, Millipore Sigma, Burlington, MA), anti-ATP8 polyclonal (mitochondrially encoded ATP8; cat. no. PA5-109987, Thermo Fisher Scientific, Waltham, MA), anti-SDHB (succinate dehydrogenase complex iron sulfur subunit B; cat. no. AP19974b, Abcepta, San Diego, CA), anti-ATP6 (mitochondrially encoded ATP6; clone no. 1G7-1G2, mAbdx, Eugene, OR), anti-CO2 (mitochondrially encoded cytochrome c oxidase II; cat. no. PA5-102933, Thermo Fisher Scientific), anti-ATP5O/OSCP (ATP synthase peripheral stalk subunit OSCP; cat. no. TA804572, Origene Technologies, Rockville, MD), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; cat. no. G9545, Sigma-Aldrich, St. Louis, MO), anti-PGK1 (phosphoglycerate kinase 1; cat. no. A14039, Abclonal, Woburn, MA), anti-GRIM19 (cat. no. ab110240, Abcam, Fremont, CA), anti-CORE-2 (ubiquinol-cytochrome c reductase core protein 2; cat. no. ab14745, Abcam), anti-ACO2 (aconitase 2; cat. no. TA500824, Origene Technologies).

Techniques: Transgenic Assay, Isolation, Western Blot, Control, Blue Native PAGE, Purification

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Exogenous expression of ATP8, a mitochondrial encoded protein, from the nucleus in vivo

doi: 10.1016/j.omtm.2024.101372

Figure Lengend Snippet: Distribution of transgenic versus endogenous ATP8 Denaturing PAGE western blots of mouse liver tissue from 6-, 12-, 30-, and 50-week-old transgenic C57BL/6J(mtC57BL/6J) mice (A left panel) and C57BL/6J(mtFVB) mice (A, right panel). The immunoblot bands of oATP8 and endogenous ATP8 were quantified by densitometry analysis (using ImageJ), normalized to GAPDH (B) and the ratio of oATP8 and endogenous ATP8 were calculated (C). Denaturing PAGE western blots of mouse liver mitochondria from non-transgenic and transgenic C57BL/6J(mtC57BL/6J) and C57BL/6J(mtFVB) mice (D). The immunoblot bands of oATP8 and endogenous ATP8 were quantified by densitometry analysis (using ImageJ), normalized to aconitase (E) and the ratio of oATP8 and endogenous ATP8 were calculated (F). Approximately fifty micrograms of protein was run per lane ( n = 3 animals per group). The animal ID alongside the gender (“M” for male and “F” for female) is listed at the top of the lanes. FLAG-tagged oATP8 protein was immunodetected with mouse anti-FLAG antibody and endogenous ATP8 was immunodetected with rabbit anti-ATP8 antibody. GAPDH and ACO2 were used as loading controls for nuclear and mitochondrial fractions. Error bars show SEM. (B) Two-way ANOVA with Šídák’s multiple comparisons: p > 0.05, ∗∗ p = 0.0018, ∗∗∗∗ p ≤ 0.0001. (C) Two-way ANOVA with Šídák’s multiple comparisons: p > 0.05, ∗ p = 0.0205, ∗∗ p = 0.00015. (E) Two-way ANOVA with Šídák’s multiple comparisons: p > 0.05, ∗ p = 0.0114. (F) Unpaired t test with Welch’s correction. p > 0.05; NS, not significant.

Article Snippet: The following antibodies were used in this study: anti-FLAG (cat. no. F1804, Millipore Sigma, Burlington, MA), anti-ATP8 polyclonal (mitochondrially encoded ATP8; cat. no. PA5-109987, Thermo Fisher Scientific, Waltham, MA), anti-SDHB (succinate dehydrogenase complex iron sulfur subunit B; cat. no. AP19974b, Abcepta, San Diego, CA), anti-ATP6 (mitochondrially encoded ATP6; clone no. 1G7-1G2, mAbdx, Eugene, OR), anti-CO2 (mitochondrially encoded cytochrome c oxidase II; cat. no. PA5-102933, Thermo Fisher Scientific), anti-ATP5O/OSCP (ATP synthase peripheral stalk subunit OSCP; cat. no. TA804572, Origene Technologies, Rockville, MD), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; cat. no. G9545, Sigma-Aldrich, St. Louis, MO), anti-PGK1 (phosphoglycerate kinase 1; cat. no. A14039, Abclonal, Woburn, MA), anti-GRIM19 (cat. no. ab110240, Abcam, Fremont, CA), anti-CORE-2 (ubiquinol-cytochrome c reductase core protein 2; cat. no. ab14745, Abcam), anti-ACO2 (aconitase 2; cat. no. TA500824, Origene Technologies).

Techniques: Transgenic Assay, Western Blot