Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Scatter plot of PCR array data showing relative gene expression levels in UTMZ cells relative to U251 cells. Genes upregulated by more than 2-fold are shown in black circles, genes downregulated by more than 2-fold are shown in black squares. Arrow shows the data point representing BMI1 . (B) Representative western blot (top) and quantitative analysis (bottom graph) showing the relative BMI1 expression levels in U251 and UTMZ cells. (C) Analysis of RNA-sequencing data provided by TCGA depicting co-expression of COX4I1 mRNA and BMI1 mRNA in patients with high-grade GBM. (D) Representative western blots depicting COX4-1 and BMI1 expression in a panel of 24 primary human GBM tumors. (E) Quantification of relative band intensities in (D) Numbers in parentheses indicate the mean value from all tumors. (F) OS for patients with high and low tumor expression levels of COX4-1 ( P < 0.0001 by the log-rank test; hazard ratio for death in patients with high tumor COX4-1 expression, 54.99; 95% CI, 11.02 to 274.3) or BMI1 ( P = 0.0113 by the log-rank test; hazard ratio for death in patients with high tumor BMI1 expression, 2.59; 95% CI, 2.107 to 3.073). Numbers in parentheses indicate the median survival time for each group.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Gene Expression, Western Blot, Expressing, RNA Sequencing

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) COX4-1 and COX4-2 constructs ( pCMV6-COX4-1-FLAG and pCMV6-COX4-2-FLAG) were transfected into U251-COX4-2 depleted cells to create U251-TgCOX4-1 and U251-TgCOX4-2 stable cell lines. Expression of COX4 isoforms and BMI1 was detected in each cell line by western blot analysis. Citrate synthase (CS) expression is shown as mitochondrial loading control and actin expression is shown as nuclear loading control. (B) Proliferation rates of each cell line. (C) Representative pictures of clonogenic assays with each cell line, showing anchorage-independent cell growth. (D) Representative images of tumors from athymic nude mice inoculated with the cell lines. Tumors were excised 4 weeks after inoculation. (E) Analysis of tumor volumes in mice over the course of the experiment. (F) Comparison of tumor weights upon excision. Graphs represent the average from triplicate determinations from at least three independent experiments.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Construct, Transfection, Stable Transfection, Expressing, Western Blot, Control, Comparison

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: Representative images of tumors resulting from intracranial implantation of U251 and U251-TgCOX4-1 glioma cells, stained for (A) H&E, (B) Ki-67, (C) COX4-1, (D) COX4-2, and (E) BMI1. Scale bar, 100 μm.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Staining

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Representative western blot depicting BMI1 expression in nuclear extracts of U251-TgCOX4-1 cell following 24-h PTC-209 treatment (0–10 μM). (B) Cell proliferation in control and PTC-209-treated (5 μM) U251-TgCOX4-1 cells. (C) Representative western blot depicting BMI1 expression in U251-TgCOX4-1 cells expressing shRNA control or one of four different vectors expressing shRNA against BMI1. (D) Quantification of the relative expression levels of BMI1 detected in (C). (E) Cell proliferation in clones expressing shRNA against BMI1. (F) Representative western blot depicting BMI1 expression levels (inset) and the cell proliferation rates of control and pCMV6-BMI1-transfected U251 cells. Graphs represent the average from triplicate determinations from at least three independent experiments.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Western Blot, Expressing, Control, shRNA, Clone Assay, Transfection

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Relative activity of CcO normalized to citrate synthase (CS) activity. (B) Oxygen consumption rates were determined using a respirometer. Representative traces of cellular respiration rates of U251 (black) and U251-TgCOX4-1 (red) cells (blue line, oxygen concentration). (C) Kinetic characterization of glutamate/malate, succinate, and fatty acid-dependent respiration of U251-TgCOX4-1 and U251-TgCOX4-2 cells. (D) Kinetic characterization of FCCP-dependent respiration in U251-TgCOX4-1 and U251-TgCOX4-2 cells. (E) Dose-response analyses of glucose uptake in cell lines expressing different COX4 isoforms. Graphs represent the average from triplicate determinations from at least three independent experiments.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Activity Assay, Concentration Assay, Expressing

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Representative histograms from flow cytometric analysis of total cellular ROS (left, DCFDA fluorescence) and mitochondrial ROS (right, MitoSOX fluorescence) in parental and U251-TgCOX4-1 cells. Bar graphs provide quantitative analysis of fluorescence intensity. (B) Quantitative graphs showing the relative levels of catalase activity, superoxide dismutase activity, NAD + /NADH ratio, and GSH/GSSG ratio in U251-TgCOX4-1 cells. (C) Representative histograms from flow cytometric analysis of total cellular or mitochondrial ROS production in U251-TgCOX4-2 cells treated with NAC (300 μM) or PTC-209 (5 μM) (left) and in U251-TgCOX4-1 cells treated with NMP (10 μM) or PTC-209 (right). (D) Representative western blots depicting BMI1 expression in the nuclear extracts of parental cells or U251-TgCOX4-1 cells after treatment with NAC or PTC-209 for 24 h (top) and quantitative analysis of expression levels (bottom). Bars represent the average from triplicate determinations from at least three independent experiments.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Fluorescence, Activity Assay, Western Blot, Expressing

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Representative phase contrast photomicrographs (10× magnification) of parental U251, U251-shRNA-COX4-2, U251-TgCOX4-1, and U251-TgCOX4-2 cells after 10 days of culture in serum-free Neurobasal medium supplemented with EGF and FGF. (B) Spheroids of U251-TgCOX4-1 cells were immunostained with antibodies against COX4-1, BMI1, or CD133 or with control antibodies. (C) In vitro limiting dilution assays and quantification of COX4-1 and BMI1 expressing cells. Results represent the average from two independent experiments. (D) Spheroid multipotency was assessed by immunofluorescence for neuronal (neurofilament, CNPase, and βIII-tubulin) and glial (GFAP) markers.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: shRNA, Control, In Vitro, Expressing, Immunofluorescence

Journal: Cell reports

Article Title: Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice

doi: 10.1016/j.celrep.2024.114284

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Deparaffinized heart tissue sections, that had been fixed with 10% formalin, were subjected to heat-induced antigen retrieval in 10 mM Tris EDTA buffer (pH 9.0) for 5 min. Antigen-retrieved sections were incubated overnight at 4°C with goat anti-CD45 antibody (R&D Systems, AF114-SP) and rabbit anti-CD68 antibody (Cell Signaling, 97778), or rabbit anti-γ-H2A.X antibody (Cell Signaling, 9718) and goat anti-desmin antibody (Invitrogen, PA5–19063), or rabbit anti-PCM1 antibody (Sigma, HPA023370), goat anti-TdTomato antibody (OriGene, AB8181), and mouse anti-Cox4 antibody (R&D Systems, MAB6980), followed by Alexa fluorophore-conjugated secondary antibodies for 1 h at 37°C.

Techniques: Recombinant, Transfection, Plasmid Preparation, Electron Microscopy, Modification, TUNEL Assay, In Situ, Generated, Cloning, Software, Imaging, Real-time Polymerase Chain Reaction, Microscopy

Journal: Cell reports

Article Title: Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics

doi: 10.1016/j.celrep.2018.10.058

Figure Lengend Snippet: The effect of COX7A2L absence on MRC complex assembly was investigated in two COX7A2L -KO clones, clone 1 (KO1) and clone 2 (KO2), compared with the control HEK293T cells (WT). (A) Mitochondria extracted with a digitonin/protein ratio of 4:1 (g/g) and analyzed by BN-PAGE, followed by CI- and CIV-IGA assays, or alternatively, by immunoblotting using the indicated antibodies. (B) Subsequent 2D-BN/SDS-PAGE and immunoblot analyses were performed with antibodies against COX7A2L and the indicated OXPHOS subunits. (C) To address the relative amount of CIII 2 in COX7A2L -KO cells, the signals from the CORE2 antibody from four BN-PAGE experiments were quantified by densitometry, normalized by CII, and indicated as mean ± SD. (D) BN-PAGE analyses in whole-cell extracts prepared in the presence of digitonin (detergent/protein ratio, 4:1) or 1% lauryl maltoside (LM). The CIII 2 signals were quantified and normalized by CII using the histogram function of the Adobe Photoshop program on digitalized images, and the values were expressed relative to the control. Error bars represent the mean ± SD of four independent experiments. (E) Spectrophotometric measurements of the individual activities of MRC complexes I to IV (CI–CIV) in WT and COX7A2L -KO cells. Enzyme activities are expressed as cU/U citrate synthase (CS). Error bars represent the mean ± SD of four repetitions. *p < 0.05; **p < 0.01. MegaC, megacomplexes probably containing more than one copy of CI, CIII 2 , and CIV. I+III 2 +IV n , SCs containing CI, CIII 2 , and CIV. I+III 2 , SC containing CI and CIII 2 . III 2 +IV, SC containing CIII 2 and CIV. III 2 , complex III dimer (CIII 2 ). IV, complex IV; IV 2 , complex IV dimer (CIV 2 ). II, complex II. Subcomplexes that contain COX1 and COX4 are indicated. Apparent subcomplexes that contain CORE2 are antibody artifacts that disappear in 2D-BN/SDS-PAGE gels. See also .

Article Snippet: COX4I1 - Myc-DDK in pCMV6-Entry , Origene , Cat# RC209374.

Techniques: Clone Assay, Control, Western Blot, SDS Page

Journal: Cell reports

Article Title: Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics

doi: 10.1016/j.celrep.2018.10.058

Figure Lengend Snippet:

Article Snippet: COX4I1 - Myc-DDK in pCMV6-Entry , Origene , Cat# RC209374.

Techniques: Recombinant, Modification, Clear Native PAGE, Software

Journal: Cell Stress

Article Title: COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM

doi: 10.15698/cst2022.04.266

Figure Lengend Snippet: (A) CcO activity in radiosensitive (U251, D456, and Jx39) and isogenic radioresistant (U251-RR, D456-RR, and Jx39-RR) cell lines. Data are presented as the mean ± SEM (n=4). (B) Representative Western blot showing the expression of the COX4 isoforms COX4-1 and COX4-2 in the radiosensitive and radioresistant cells. COX-I was probed as a loading control. (C) Digitonin-solubilized mitochondria from radiosensitive and radioresistant cell lines were subjected to BN-PAGE followed by complex I and IV IGA assays and Western blot for complex III. Representative image from 3 separate experiments. p < 0.0001 (****), calculated by Student t-test.

Article Snippet: Glioma cells were electroporated with a lentiviral vector carrying one of four unique 29mer shRNA constructs specific for human COX4I1 or scramble control shRNA (OriGene Technologies, Rockville, MD; catalog # TL313764).

Techniques: Activity Assay, Western Blot, Expressing, Control

Journal: Cell Stress

Article Title: COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM

doi: 10.15698/cst2022.04.266

Figure Lengend Snippet: (A) Representative Western blot showing COX4-1 expression in U251 and D456 cells stably transfected with vector only or vector expressing COX4-1. Citrate synthase (CS) served as the loading control. (B) Quantification of CcO activity in vector control- and COX4-1-transfected U251 and D456 cells. Data are presented as the mean ± SEM (n=4). (C) Digitonin-solubilized mitochondria from the indicated cell lines were subjected to BN-PAGE followed by in-gel complex I and IV activity assays and complex III Western blot analysis. Representative images from 3 separate experiments. p < 0.0001 (****), calculated by Student t-test.

Article Snippet: Glioma cells were electroporated with a lentiviral vector carrying one of four unique 29mer shRNA constructs specific for human COX4I1 or scramble control shRNA (OriGene Technologies, Rockville, MD; catalog # TL313764).

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Activity Assay

Journal: Cell Stress

Article Title: COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM

doi: 10.15698/cst2022.04.266

Figure Lengend Snippet: Quantification of mitochondrial O 2 •- production in vector-transfected controls and COX4-1-overexpressing U251 cells (A) and D456 (B) cells treated with or without AA (10 μM for 15 min), assessed by MitoSOX assay. Data are presented as the mean ± SEM (n= 3). Clonogenic survival curves for vector-transfected controls and COX4-1-overexpressing U251 (C) and D456 (D) cells. Cells were irradiated with 2, 4, 6, or 8 Gy and immediately plated. Clonogenic survival was estimated on day 14 after irradiation. p < 0.01 (**) and p < 0.001 (***), calculated using one-way ANOVA followed by Tukey's multiple comparison test. p < 0.0001 (****), calculated by Student t-test. a.u., arbitrary units; AA, antimycin A; ns, not significant.

Article Snippet: Glioma cells were electroporated with a lentiviral vector carrying one of four unique 29mer shRNA constructs specific for human COX4I1 or scramble control shRNA (OriGene Technologies, Rockville, MD; catalog # TL313764).

Techniques: Plasmid Preparation, Transfection, Mitosox Assay, Irradiation, Comparison

Journal: Cell Stress

Article Title: COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM

doi: 10.15698/cst2022.04.266

Figure Lengend Snippet: (A) Representative Western blot showing COX4-1 expression in U251-RR and D456-RR cells transfected with shRNA targeting COX4-1 or scramble shRNA. Citrate synthase (CS) served as the loading control. (B) Quantification of CcO activity in in U251-RR and D456-RR cells transfected with shRNA targeting COX4-1 or scramble shRNA. Data are presented as the mean ± SEM (n=3) (C) Digitonin-solubilized mitochondria from the indicated cell lines were subjected to BN-PAGE followed by complex I and IV IGA assays and complex III Western blot analysis. Representative images from 4 independent preparations. p < 0.0001 (****), calculated by Student t-test.

Article Snippet: Glioma cells were electroporated with a lentiviral vector carrying one of four unique 29mer shRNA constructs specific for human COX4I1 or scramble control shRNA (OriGene Technologies, Rockville, MD; catalog # TL313764).

Techniques: Western Blot, Expressing, Transfection, shRNA, Control, Activity Assay

Journal: Cell Stress

Article Title: COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM

doi: 10.15698/cst2022.04.266

Figure Lengend Snippet: (A) Quantification of mitochondrial O 2 •- production in U251-RR (top) and D456-RR (bottom) cells transfected with shRNA targeting COX4-1 or scramble shRNA and treated with or without AA (10 μM for 15 min), assessed by MitoSOX assay. Data are presented as the mean ± SEM (n=3). (B) Clonogenic survival curves for U251-RR (top) and D456-RR (bottom) cells transfected with shRNA targeting COX4-1 or scramble shRNA. Cells were irradiated (0–8 Gy) and immediately plated. Clonogenic survival was estimated on day 14 after irradiation. ***p<0.001, calculated using one-way ANOVA followed by Tukey's multiple comparison test. p < 0.0001 (****), calculated by Student t-test. a.u., arbitrary units; AA, antimycin A; ns, not significant.

Article Snippet: Glioma cells were electroporated with a lentiviral vector carrying one of four unique 29mer shRNA constructs specific for human COX4I1 or scramble control shRNA (OriGene Technologies, Rockville, MD; catalog # TL313764).

Techniques: Transfection, shRNA, Mitosox Assay, Irradiation, Comparison

Journal: Cell Stress

Article Title: COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM

doi: 10.15698/cst2022.04.266

Figure Lengend Snippet: (A) CcO activity in human GBM tumors. Data are presented as the mean ± SEM (n=3). (B) Survival time of patients. (C) Representative Western blot showing the expression of COX4-1 in GBM tumors. Citrate synthase (CS) was probed as a loading control. (D) Densitometric analysis of Western blot in C. (E) Digitonin-solubilized mitochondria from GBM tumors were subjected to BN-PAGE followed by complex I and IV IGA assays and complex III Western blot analysis.

Article Snippet: Glioma cells were electroporated with a lentiviral vector carrying one of four unique 29mer shRNA constructs specific for human COX4I1 or scramble control shRNA (OriGene Technologies, Rockville, MD; catalog # TL313764).

Techniques: Activity Assay, Western Blot, Expressing, Control