primary anti cox iv  (Proteintech)

Journal: Diabetes & Metabolism Journal

Article Title: Targeting SLC25A33 Suppresses Vascular Smooth Muscle Cell Proliferation and Migration by Reducing Cytosolic mtDNA Levels: Implications for Occlusive Vascular Diseases

doi: 10.4093/dmj.2024.0632

Figure Lengend Snippet: Solute carrier family 25 member 33 (SLC25A33) knockdown in vascular smooth muscle cells (VSMCs) reduces neointima formation in a carotid artery ligation model. (A) Representative images of hematoxylin and eosin (H&E) staining (upper panel; scale bar=50 μm), indicating the intimal and medial layers, as well as the intima/media ratio (lower panel). (B, C) Immunohistochemical staining with antibodies against SLC25A33, phosphorylated p65 (p-p65), and phosphorylated TANK-binding kinase 1 (p-TBK1) in mouse carotid artery tissue sections (B) and quantification of positive staining for SLC25A33, p-p65, and p-TBK1 (C). (D, E) Representative immunofluorescence images of cytochrome c oxidase (COX) IV and 8-hydroxy-2’-deoxyguanosine (8-OHdG) (D) and quantification of cytosolic 8-OHdG in mouse carotid artery tissue sections (E). COX IV protein is used as a mitochondrial marker. sh SLC25A33 is referred to as shA33. Data in the bar graphs represent the mean±standard error of the mean of six independent animals. Scale bar=10 μm. NS, not significant; DAPI, 4’,6-diamidino-2-phenylindole. a P <0.05, b P <0.01, c P <0.001.

Article Snippet: Primary antibodies against cytochrome c oxidase (COX) IV (Cell Signaling) and 8-hydroxy-2 ́-deoxyguanosine (8-OHdG) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for immunostaining.

Techniques: Knockdown, Ligation, Staining, Immunohistochemical staining, Binding Assay, Immunofluorescence, Marker

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Nuclear Control of Mitochondrial Homeostasis and Venetoclax Efficacy in AML via COX4I1.

doi: 10.1002/advs.202404620

Figure Lengend Snippet: Figure 1. Identification of the essential role of COX4I1 in leukemia cells through cell signaling CRISPR screens. A) Schematic representation of the design and cloning process of the cell signaling CRISPR library. B) Volcano plot illustrating the log2 fold change of sgRNA abundance over 28 days of screen culture (x-axis; log2FC) and the significance (y-axis; MAGeCK score) of each gene in the cell signaling CRISPR screen of Molm13-Cas9+ cells. Essential genes are highlighted in red, negative controls in blue, and the total library in gray. C) Dot plots showing the CRISPR gene dependency (CERE) score of COX4I1 across 1021 human cancer cell lines tested in the DepMap consortium (BROAD Institute). D) Expression profile of COX4I1 in normal

Article Snippet: PVDF membranes were subsequently blocked with 5% bovine serum albumin (Fisher Scientific) in Tris-buffer saline-tween 20 (TBST) at room temperature for 1 h. Primary antibodies targeting COX4I1 (rabbit mAb [clone 3E11]; 4850, Cell Signaling Technology, 1:1000), OPA1 (rabbit mAb [clone D6U6N]; 80471, Cell Signaling Technology, 1:1000), cytochrome c (rabbit mAb [clone EPR1327]; ab133504, Abcam, 1:1000), VDAC (rabbit mAb [clone D73D12]; 4661, Cell Signaling Technology, 1:1000), COX5A (rabbit mAb [clone EPR14208(B)]; ab180129, Abcam, 1:1000), MT-CO2 (rabbit mAb [clone E6U9K]; 50003, Cell Signaling Technology, 1:1000), COX6B1 (mouse mAb [clone 3F9D3D11AF6]; ab110266, Abcam, 1:500), cleaved caspase 3 (rabbit mAb [clone 5A1E]; 9664, Cell Signaling Technology, 1:1000), BAX (rabbit mAb [clone D2E11], 5023, Cell Signaling Technology, 1:1000), BAK (rabbit mAb [clone D4E4], 12105, Cell Signaling Technology, 1:1000), and β-actin (mouse mAb [clone 8H10D10]; 3700, Cell Signaling Technology; 1:1000) were then applied to the membranes at 4 °C overnight.

Techniques: CRISPR, Cloning, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Nuclear Control of Mitochondrial Homeostasis and Venetoclax Efficacy in AML via COX4I1.

doi: 10.1002/advs.202404620

Figure Lengend Snippet: Figure 2. COX4I1 is indispensable for in vivo AML progression. A) Schematic representation of a “human-in-mouse” leukemia xenograft model and in vivo bioluminescent images using NSGS mice injected with Cas9/luciferase-expressing (Cas9+/Luc+) human Molm13 AML cells transduced with sgCtrl and sgCOX4I1 (n = 7 mice per group). B) Quantitative analysis of bioluminescent signals from NSGS recipient mice transplanted with Molm13- Cas9+/Luc+ AML cells transduced with sgCtrl and sgCOX4I1 (n = 7 mice per group). C,D) Impact of COX4I1 depletion on (C) spleen weight and (D) spleen size (n = 4 mice per group). E,F) Representative flow cytometric profiles of RFP and human CD45 (hCD45) expression (E) and the percentage of RFP+/hCD45+ human AML cells in the spleen (n = 3) and peripheral blood (n = 5) of mice receiving Molm13-Cas9+/Luc+ AML cells transduced with sgCtrl and sgCOX4I1 (F). G) Kaplan-Meier survival curve of NSGS recipient mice transplanted with Molm13-Cas9+/Luc+ AML cells transduced with sgCtrl and sgCOX4I1 (n = 7 mice per group). Data are presented as mean ± SEM. *p < 0.01 by two-sided Student’s t-test.

Article Snippet: PVDF membranes were subsequently blocked with 5% bovine serum albumin (Fisher Scientific) in Tris-buffer saline-tween 20 (TBST) at room temperature for 1 h. Primary antibodies targeting COX4I1 (rabbit mAb [clone 3E11]; 4850, Cell Signaling Technology, 1:1000), OPA1 (rabbit mAb [clone D6U6N]; 80471, Cell Signaling Technology, 1:1000), cytochrome c (rabbit mAb [clone EPR1327]; ab133504, Abcam, 1:1000), VDAC (rabbit mAb [clone D73D12]; 4661, Cell Signaling Technology, 1:1000), COX5A (rabbit mAb [clone EPR14208(B)]; ab180129, Abcam, 1:1000), MT-CO2 (rabbit mAb [clone E6U9K]; 50003, Cell Signaling Technology, 1:1000), COX6B1 (mouse mAb [clone 3F9D3D11AF6]; ab110266, Abcam, 1:500), cleaved caspase 3 (rabbit mAb [clone 5A1E]; 9664, Cell Signaling Technology, 1:1000), BAX (rabbit mAb [clone D2E11], 5023, Cell Signaling Technology, 1:1000), BAK (rabbit mAb [clone D4E4], 12105, Cell Signaling Technology, 1:1000), and β-actin (mouse mAb [clone 8H10D10]; 3700, Cell Signaling Technology; 1:1000) were then applied to the membranes at 4 °C overnight.

Techniques: In Vivo, Injection, Luciferase, Expressing, Transduction

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Nuclear Control of Mitochondrial Homeostasis and Venetoclax Efficacy in AML via COX4I1.

doi: 10.1002/advs.202404620

Figure Lengend Snippet: Figure 3. COX4I1 is crucial for maintaining mitochondrial homeostasis. A) RNA-seq and Gene Set Enrichment Analysis (GSEA) revealing alterations in the expression of “E2F Targets” and “Mitochondrial Stress” gene sets in sgCtrl versus sgCOX4I1-transduced Molm13-Cas9+ cells. Each dot repre- sents a Hallmark gene set from the GSEA Molecular Signature Database. NES indicates normalized enrichment score. B) Representative images from transmission electron microscopy depicting mitochondrial ultrastructure and C) measurement of maximal cristae width observed in Molm13-Cas9+

Article Snippet: PVDF membranes were subsequently blocked with 5% bovine serum albumin (Fisher Scientific) in Tris-buffer saline-tween 20 (TBST) at room temperature for 1 h. Primary antibodies targeting COX4I1 (rabbit mAb [clone 3E11]; 4850, Cell Signaling Technology, 1:1000), OPA1 (rabbit mAb [clone D6U6N]; 80471, Cell Signaling Technology, 1:1000), cytochrome c (rabbit mAb [clone EPR1327]; ab133504, Abcam, 1:1000), VDAC (rabbit mAb [clone D73D12]; 4661, Cell Signaling Technology, 1:1000), COX5A (rabbit mAb [clone EPR14208(B)]; ab180129, Abcam, 1:1000), MT-CO2 (rabbit mAb [clone E6U9K]; 50003, Cell Signaling Technology, 1:1000), COX6B1 (mouse mAb [clone 3F9D3D11AF6]; ab110266, Abcam, 1:500), cleaved caspase 3 (rabbit mAb [clone 5A1E]; 9664, Cell Signaling Technology, 1:1000), BAX (rabbit mAb [clone D2E11], 5023, Cell Signaling Technology, 1:1000), BAK (rabbit mAb [clone D4E4], 12105, Cell Signaling Technology, 1:1000), and β-actin (mouse mAb [clone 8H10D10]; 3700, Cell Signaling Technology; 1:1000) were then applied to the membranes at 4 °C overnight.

Techniques: RNA Sequencing, Expressing, Transmission Assay, Electron Microscopy

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Nuclear Control of Mitochondrial Homeostasis and Venetoclax Efficacy in AML via COX4I1.

doi: 10.1002/advs.202404620

Figure Lengend Snippet: Figure 4. COX4I1 regulates mitochondrial respiration and Complex IV formation. A) Oxygen consumption rate (OCR) measured in Cas9-expressing Molm13 (left), MV4-11 (middle), and THP-1 (right) cells transduced with sgCtrl (gray; n = 3 independent sgRNA sequences) and sgCOX4I1 (red; n = 3 independent sgRNA sequences). (B and C) Relative levels of AMP, ADP, and ATP in B) Molm13-Cas9+ cells transduced with sgCtrl versus sgCOX4I1, and C) COX4I1-knockout (KO) Molm13 cells transduced with an empty vector (Vec) versus a synthetic human COX4I1 cDNA. D) Schematic representation of mitochondrial oxidative phosphorylation comprising electron transport chain complexes (I – IV) and ATP synthase (V). H+ denotes a proton, and e–

Article Snippet: PVDF membranes were subsequently blocked with 5% bovine serum albumin (Fisher Scientific) in Tris-buffer saline-tween 20 (TBST) at room temperature for 1 h. Primary antibodies targeting COX4I1 (rabbit mAb [clone 3E11]; 4850, Cell Signaling Technology, 1:1000), OPA1 (rabbit mAb [clone D6U6N]; 80471, Cell Signaling Technology, 1:1000), cytochrome c (rabbit mAb [clone EPR1327]; ab133504, Abcam, 1:1000), VDAC (rabbit mAb [clone D73D12]; 4661, Cell Signaling Technology, 1:1000), COX5A (rabbit mAb [clone EPR14208(B)]; ab180129, Abcam, 1:1000), MT-CO2 (rabbit mAb [clone E6U9K]; 50003, Cell Signaling Technology, 1:1000), COX6B1 (mouse mAb [clone 3F9D3D11AF6]; ab110266, Abcam, 1:500), cleaved caspase 3 (rabbit mAb [clone 5A1E]; 9664, Cell Signaling Technology, 1:1000), BAX (rabbit mAb [clone D2E11], 5023, Cell Signaling Technology, 1:1000), BAK (rabbit mAb [clone D4E4], 12105, Cell Signaling Technology, 1:1000), and β-actin (mouse mAb [clone 8H10D10]; 3700, Cell Signaling Technology; 1:1000) were then applied to the membranes at 4 °C overnight.

Techniques: Expressing, Transduction, Knock-Out, Plasmid Preparation, Phospho-proteomics

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Nuclear Control of Mitochondrial Homeostasis and Venetoclax Efficacy in AML via COX4I1.

doi: 10.1002/advs.202404620

Figure Lengend Snippet: Figure 5. COX4I1 governs pivotal stages of Complex IV assembly. A) Schematic depiction of the COX4I1 high-resolution CRISPR tiling library screen conducted in Molm13-Cas9+ cells. B) 2D annotation illustrating COX4I1 peptide position (x-axis) and CRISPR tiling score (y-axis). The red line represents the smoothed model of the CRISPR scan score derived from individual sgRNAs (dots) targeting the coding exons of COX4I1. The CRISPR hypersensitive P1 and P2 regions are indicated. C,D) Impact of wild-type (WT), ΔP1, and ΔP2 COX4I1 expression on (C) cell number expansion and (D) mitochon- drial respiration in COX4I1-KO Molm13 cells (n = 3 for each group). E) Western blot analysis of COX4I1, COX5A, MT-CO2, COX6B1, and 𝛽-actin in

Article Snippet: PVDF membranes were subsequently blocked with 5% bovine serum albumin (Fisher Scientific) in Tris-buffer saline-tween 20 (TBST) at room temperature for 1 h. Primary antibodies targeting COX4I1 (rabbit mAb [clone 3E11]; 4850, Cell Signaling Technology, 1:1000), OPA1 (rabbit mAb [clone D6U6N]; 80471, Cell Signaling Technology, 1:1000), cytochrome c (rabbit mAb [clone EPR1327]; ab133504, Abcam, 1:1000), VDAC (rabbit mAb [clone D73D12]; 4661, Cell Signaling Technology, 1:1000), COX5A (rabbit mAb [clone EPR14208(B)]; ab180129, Abcam, 1:1000), MT-CO2 (rabbit mAb [clone E6U9K]; 50003, Cell Signaling Technology, 1:1000), COX6B1 (mouse mAb [clone 3F9D3D11AF6]; ab110266, Abcam, 1:500), cleaved caspase 3 (rabbit mAb [clone 5A1E]; 9664, Cell Signaling Technology, 1:1000), BAX (rabbit mAb [clone D2E11], 5023, Cell Signaling Technology, 1:1000), BAK (rabbit mAb [clone D4E4], 12105, Cell Signaling Technology, 1:1000), and β-actin (mouse mAb [clone 8H10D10]; 3700, Cell Signaling Technology; 1:1000) were then applied to the membranes at 4 °C overnight.

Techniques: CRISPR, Derivative Assay, Expressing, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Nuclear Control of Mitochondrial Homeostasis and Venetoclax Efficacy in AML via COX4I1.

doi: 10.1002/advs.202404620

Figure Lengend Snippet: Figure 6. Targeting COX4I1 sensitizes AML cells to venetoclax treatment. A) Impact of targeting COX4I1 on leukemia cells treated with azacytidine, cytarabine, and venetoclax. B) Effect of targeting COX4I1 on protein levels of COX4I1, cleaved caspase 3 (c-Cas3), and 𝛽-actin in leukemia cells treated with venetoclax. C) Representative flow cytometric profiles of mitochondrial membrane potential (MPP), and D) the effect of targeting COX4I1 on venetoclax-induced mitochondrial depolarization (n = 3 for each group). E) Western blot analysis of BAX, BAK, and 𝛽-actin in leukemia cells transduced with sgLuc/Ren and sgBAX/BAK. F) Effect of targeting COX4I1 on venetoclax-induced apoptosis in leukemia cells transduced with sgLuc/Ren and sgBAX/BAK (n = 3 for each group). G) Schematic model illustrating the enhancement of venetoclax-induced apoptosis through COX4I1 targeting. Data are presented as mean ± SEM. *p < 0.01 by two-sided Student’s t-test.

Article Snippet: PVDF membranes were subsequently blocked with 5% bovine serum albumin (Fisher Scientific) in Tris-buffer saline-tween 20 (TBST) at room temperature for 1 h. Primary antibodies targeting COX4I1 (rabbit mAb [clone 3E11]; 4850, Cell Signaling Technology, 1:1000), OPA1 (rabbit mAb [clone D6U6N]; 80471, Cell Signaling Technology, 1:1000), cytochrome c (rabbit mAb [clone EPR1327]; ab133504, Abcam, 1:1000), VDAC (rabbit mAb [clone D73D12]; 4661, Cell Signaling Technology, 1:1000), COX5A (rabbit mAb [clone EPR14208(B)]; ab180129, Abcam, 1:1000), MT-CO2 (rabbit mAb [clone E6U9K]; 50003, Cell Signaling Technology, 1:1000), COX6B1 (mouse mAb [clone 3F9D3D11AF6]; ab110266, Abcam, 1:500), cleaved caspase 3 (rabbit mAb [clone 5A1E]; 9664, Cell Signaling Technology, 1:1000), BAX (rabbit mAb [clone D2E11], 5023, Cell Signaling Technology, 1:1000), BAK (rabbit mAb [clone D4E4], 12105, Cell Signaling Technology, 1:1000), and β-actin (mouse mAb [clone 8H10D10]; 3700, Cell Signaling Technology; 1:1000) were then applied to the membranes at 4 °C overnight.

Techniques: Membrane, Western Blot, Transduction