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

Journal: Metabolites

Article Title: Effect of Expression of Nuclear-Encoded Cytochrome C Oxidase Subunit 4 Isoforms on Metabolic Profiles of Glioma Cells

doi: 10.3390/metabo12080748

Figure Lengend Snippet: Metabolite profiling of U251 glioma cells overexpressing the COX4-1 or COX4-2 isoform. ( A ) Two-dimensional PCA score plots of untargeted metabolomics data from COX4-1-overexpressing glioma cells (blue) and COX4-2-overexpressing glioma cells (red). ( B ) Volcano plot of metabolomic data. The x-axis represents the mean fold change (log 2 ratio) in the relative intensity of each metabolite between the two samples (COX4-2-overexpressing relative to the COX4-1-overexpressing cells). The y-axis represents the statistical significance (-log 10 -transformed p -values) of each metabolite. Metabolites are color coded by metabolic pathway.

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

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

doi: 10.1101/2023.03.20.533453

Figure Lengend Snippet: ( A ) 8-3 staining after siRNA knockdown of ACTB in MiaPaca2 cells, co-stained with phalloidin, MYH10 antibody and DAPI. ( B ) Measurement of 19-3 binding to recombinant RUVBL1 and RUBL2 proteins by ELISA. Positive controls using polyclonal rabbit antibodies anti-RUVBL1 (a-RUVBL1) or anti-RUVBL2 (a-RUVBL2). ( C ) 19-3 staining after siRNA knockdown of RUVBL2 in MiaPaca2 cells, co-stained with anti-RUVBL2 rabbit antibody and DAPI. ( D ) Co-staining of 15-7 with mitochondria markers COX4I1, HSPA9 and HSPD1 in MiaPaca2 cells. Scale bars in A, C and D are 50um.

Article Snippet: Other antibodies and dyes used were: Phalloidin (Thermo Fisher, #A12379), MYH10 (Atlas Antibodies, #HPA047541), cytochalasin D (Thermo Fisher, #PHZ1063), ACTIN (Cell Signaling Technology, #3700S), RUVBL2 (Atlas Antibodies, #HPA067966), RUVBL1 (Atlas Antibodies, # HPA019947), HSPD1 (Atlas Antibody, #HPA050025), HSPA9 (Atlas Antibody, #HPA000898), COX4I1 (Atlas antibody, #HPA002485), KRT19 (Abcam, #ab203445), human IgG isotype controls (Biolegend, #403502 and #403702).

Techniques: Staining, Knockdown, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Lactate mitochondrial oxidation drives stemness potential in metastatic breast cancer

doi: 10.1038/s41467-025-67091-y

Figure Lengend Snippet: A , B Schematic of 2- 2 H-lactate metabolism ( A ) and 2 H-labeled M + 1 metabolites ( B , n = 3 biologically independent samples). ** P = 0.0018, **** P < 0.0001; two-tailed t test. C , D CD147/MCT1/LDHB protein levels in mitochondria ( C ) and sub-compartments ( D ) from AR cells. K proteinase K, T triton X-100, O/I outer membrane and intermembrane space, I/M inner membrane and matrix. COX IV, Na + -K + ATPase, α-Tubulin, TOMM20 mark of inner membrane, plasma membrane, cytoplasm, outer membrane. Data are representative of 3 independent experiments. E Transmission electron microscopy shows CD147/MCT1/LDHB co-localization (orange arrows) in the mitochondrial inner membrane of AR cells. Scale bars: 1 μm (left), 500 nm (right). Red asterisks, mitochondria. F Proximity ligation assay (PLA) between CD147 and LDHB in lactate-treated MCF7 cells. Scale bars, 5 μm. n = 6 typical fields from 3 biologically independent samples. DAPI (blue), PLA signal (red). **** P < 0.0001; two-tailed t test. G Pull-down assay between GST-CD147 N-terminal and His-LDHB. Representative of 3 independent experiments. H CD147 (red)/LDHB (blue)/MCT1 (purple) co-localization (orange arrows) in COX IV (green) labeled mitochondria from PDX1 lung metastatic tumors. Scale bars: 20 μm. I CD147 (green)/LDHB (yellow)/MCT1 (orange) expressions in CK18 (red) labeled cells from breast cancer patients with or without metastasis. Scale bars: 40 μm. J Correlation between CD147 and MCT1/LDHB in 11 metastatic patients with breast cancer. R 2 , Pearson correlation coefficient. Spearman correlation analysis. K Kaplan–Meier survival (up) and recurrence (down) analysis for 131 patients stratified by CD147/MCT1/LDHB co-expression (high n = 9 patients, low n = 31 patients). L Representative images of HE (left: scale bars, 50 μm) and SOX2 IHC staining (middle: scale bars, 50 μm ; right : enlarged image, scale bars, 10 μm) in CTCs from No. 9 patient in Fig. . M Co-localization (orange arrows) of CD147 (red)/LDHB (blue)/MCT1 (purple) in COX IV (green) labeled mitochondria of CTCs from No. 9 patient in Fig. . Scale bars: 20 μm. N SOX2 IHC intensity in CTCs from 10 patients stratified by mitochondrial CD147/MCT1/LDHB complex presence (without, n = 4 patients; with, n = 6 patients). *** P = 0.0004, two-tailed t test. Data are mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: For cellular co-localization analysis among CD147, MCT1, LDHB, and COX IV, cells were initially stained with primary antibodies against CD147 (1:200), MCT1 (1:200; 20139-1-AP, Proteintech), and LDHB (1:100; ab195964, Abcam) overnight at 4 °C, followed by incubation with Alexa Fluor 555-, 647- or 405-conjugated antibodies, respectively, at room temperature for 1 h. This was followed by incubation with an Alexa Fluor 488-conjugated COX IV antibody (1:200; CL488-60251, Proteintech) at room temperature for 3–4 h. Finally, nuclei were counterstained, and images were captured following the same procedures as described above.

Techniques: Labeling, Two Tailed Test, Membrane, Clinical Proteomics, Transmission Assay, Electron Microscopy, Proximity Ligation Assay, Pull Down Assay, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Lactate mitochondrial oxidation drives stemness potential in metastatic breast cancer

doi: 10.1038/s41467-025-67091-y

Figure Lengend Snippet: A Immunoactivity of EGFP-fused mito-CD147 scFv to CD147 in MCF7/T47D cell subfractions: C (plasma membrane and cytoplasm), M (mitochondria), COX IV, α-Tubulin mark mitochondrial inner membrane, cytoplasm. NC: pGV469 vector; scFv: pGV469-mito-CD147 scFv. Representative of 3 independent experiments. B Co-IP assays showing CD147 interaction with MCT1/LDHB in mitochondria from lactate-treated MCF7 WT/KO/mito147 cells as indicated. Groups: 1. WT; 2. WT+mito-CD147 scFv; 3. KO; 4. KO+mito-CD147 scFv; 5. mito147; 6. mito147+mito-CD147 scFv. Representative of 3 independent experiments. C–F Experimental design ( C ), lung nodules ( D , n = 10 mice/group), ALDH + and CD44 high CD24 −/low ratios ( E , n = 5 mice/group), and SOX2 levels ( F , n = 4 mice/group, α-Tubulin, loading control) in tumors from MMTV-PyMT mice as indicated. *** P = 0.0001 ( E left), *** P = 0.0004, 0.0003 ( E right, NS vs Lac, Lac vs Lac + scFv), **** P < 0.0001, one-way ANOVA with Bonferroni correction. C created in BioRender. Song, C. (2025) https://BioRender.com/37z58my . G , H CTC counts ( G , left, n = 8 mice/group), SOX2 intensity ( G , right; NS, n = 148 cells from 7 mice; Lac, n = 342 cells from 7 mice; Lac+scFv, n = 149 cells from 7 mice), and representative images of CTCs ( H ; scale bars, 40 μm), from MMTV-PyMT mice as indicated. H DAPI (blue), CK18 (green), SOX2 (purple). **** P < 0.0001, one-way ANOVA with Bonferroni correction. I , J Lung nodules ( I , n = 8 mice/group), ALDH + and CD44 high CD24 −/low ratios ( J , n = 5 mice/group) in tumors from PDX1-bearing mice as indicated. ns, P = 0.9818, 0.869 ( J left, right), *** P = 0.0001, **** P < 0.0001, one-way ANOVA with Bonferroni correction. K–M CTC counts ( K , left, n = 8 mice/group), SOX2 intensity ( K , right; NS, n = 80 cells from 8 mice; scFv, n = 80 cells from 8 mice; Lac, n = 120 cells from 6 mice; Lac+scFv, n = 80 cells from 8 mice), representative CTC images ( L ), and mito-CD147 scFv binding to mitochondrial CD147 ( M ) in CTCs from PDX1-bearing mice as indicated. Scale bars: 10 μm. L DAPI (blue), CK18 (green), SOX2 (purple). M DAPI (blue), CD147 (red), scFv (green), COX IV (purple). CTC circulating tumor cell, scFv single-chain variable fragment. Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: For cellular co-localization analysis among CD147, MCT1, LDHB, and COX IV, cells were initially stained with primary antibodies against CD147 (1:200), MCT1 (1:200; 20139-1-AP, Proteintech), and LDHB (1:100; ab195964, Abcam) overnight at 4 °C, followed by incubation with Alexa Fluor 555-, 647- or 405-conjugated antibodies, respectively, at room temperature for 1 h. This was followed by incubation with an Alexa Fluor 488-conjugated COX IV antibody (1:200; CL488-60251, Proteintech) at room temperature for 3–4 h. Finally, nuclei were counterstained, and images were captured following the same procedures as described above.

Techniques: Clinical Proteomics, Membrane, Plasmid Preparation, Co-Immunoprecipitation Assay, Control, Binding Assay