alkaline phosphatase conjugated streptavidin biotin abc kit  (Vector Laboratories)

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Cox4i2 deficiency reduces inflammation in mice after 8 months of cigarette smoke exposure. a-d) FACS analysis of bronchoalveolar lavage (BAL) from mice exposed to cigarette smoke (CS) or room air (RA) for 8 months ( n =4 per group): Numbers of neutrophils (a), CD3 + T cells (b), CD4 + T cells (c) and CD8 + T cells (d). e-h) Immunofluorescence analysis of CD45 + (e, f) and CD3 + (g, h) cell accumulation in the lung parenchyma of mice exposed to CS or RA for 8 months ( n =4 per group). e, g: Quantification. f, h: Representative images. i-l) Microarray analysis of lung homogenates from mice exposed to CS or RA for 8 months ( n =6 per group). Most differentially regulated pathways between Cox4i2 −/− and WT mice (i). Differentially expressed genes involved in inflammatory pathways: NF-κB signalling (j), cytokine–cytokine receptor interaction (k) and T-cell receptor signalling (l). m) Multiplex analysis of BAL fluid (BALF) from mice exposed to CS or RA for 8 months ( n =5 per group). Statistical analysis was performed using two-way ANOVA. Data from panels a-e, g and m were log-transformed prior to statistical analysis. The data are presented as the mean ± SEM.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Immunofluorescence, Microarray, Multiplex Assay, Transformation Assay

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Cox4i2 is predominantly expressed in pericytes, smooth muscle cells and fibroblasts a) Schematic illustration of sample acquisition and data generation from CD45 + and CD45 - cells for single-cell RNAseq analyses. b) UMAP representation of CD45 - cells ( n =2–3 per group). c) UMAP highlighting Cox4i2 expressing cells in the pericyte/ smooth muscle cells (SMC) cluster of CD45 - cells. d) DotPlot visualization of marker genes for pericytes, SMCs and fibroblasts (FBs). e) Protein expression of COX4I2 in primary lung FBs, pulmonary artery SMCs (PASMCs), alveolar type II cells (ATIIs), endothelial cells (ECs), pericyte pellet, T cells, neutrophils and macrophages. PASMCs isolated from Cox4i2 −/− mice served as a negative control. f) Track plot of pericyte markers and fibroblast markers g) UMAP representation of the expression of Cox4i2 , Acta2 , or both in pericytes. Red: cells expressing Acta2 ; green: cells expressing Cox4i2 ; yellow: cells expressing both Acta2 and Cox4i2 . h) DotPlot visualization of marker genes expressed in pericytes. i) Labelling of Cox4i2 -expressing cells was performed using Cox4i2 -CreERT2-tdTomato mice. A Kozak-CreERT2-P2A cassette was inserted just before the Cox4i2 start codon, and these mice were crossed with tdTomato reporter mice containing a loxP-STOP-loxP cassette. After tamoxifen (i.p.; TAM) injection, Cre activation removed the stop cassette, enabling tdTomato expression driven by the Cox4i2 promoter. j, k) Representative images. j: tdTomato-positive cells in alveolar structures costained with CD31 as an endothelial marker and ACTA2 as an SMC marker. k: tdTomato-positive cells in pulmonary vessels. Colour code: Red – tdTomato, green – ACTA2, bright cyan – CD31, blue – Hoechst.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Single Cell, RNA sequencing, Expressing, Marker, Isolation, Negative Control, Injection, Activation Assay

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Cell clusters identified by scRNA-seq of CD45 - and CD45 + lung cells after 3 months of CS exposure a) UMAP representation of CD45 + cells (n=2–3 per group). b, c) Numbers of different clusters of CD45 - (b) and CD45 + (c) cells.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Cell marker genes of CD45 - and CD45 + lung clusters a) DotPlot visualization of the gene expression of different cell markers in the CD45 - clusters. b) DotPlot visualization of the gene expression of different cell markers in the CD45 + clusters.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Marker, Gene Expression

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Expression of Cox4i2 in different cell clusters identified by scRNA-seq of CD45 - and CD45 + lung cells a-c) Annotation strategy of neutrophils at different developmental stages (a) by analysing pseudotime trajectory (b) and violin plots of gene expression of different neutrophil markers (c): Cd101 + neutrophils ( Cd101, Csf3r, and Il1b ), Ngp + neutrophils ( Ngp, Ltf, and Camp ), and Treml4+ neutrophils ( Treml4, Cx3cr1, and Clec4a1 ). d-f) Schematic illustration of the developmental stage of monocytes (d); pseudotime trajectory of monocytes (e) and violin plots of monocyte and macrophage markers (f). Monocyte/macrophage clusters expressing the classical marker Cd68 were classified on the basis of distinct marker expression: alveolar macrophages ( Plet1, Lpl, and Hebp1 ), interstitial macrophages ( C1qa, C1qb, and C1qc ), Cd300e + macrophages ( Ace, Cd300e, and Dusp16 ), monocytes ( Fn1, F13a1, and Ccr2 ), and transitional monocyte/macrophage clusters ( Nr4a1, Apoc2, and Clec4a1 ), which coexpress markers of nonclassical monocytes and maladaptive macrophages. g) Expression of Cox4i2 mRNA across different cell clusters within CD45 - and CD45 + cell populations.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Expressing, Gene Expression, Marker

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet:

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: a) WT mice were exposed to room air (RA) or cigarette smoke (CS) for 8 months, followed by treatment with MitoQ (50 mg/kg/day) or the inactive carrier decylTPP+ for 3 months via gavage. b) Lung functional analyses ( n =7-8 per group) of static compliance. c-d) Stereological analysis of the lung parenchyma ( n =6 per group) showing the number of alveoli (c) and representative pictures (d). e) Haemodynamic measurements ( n =8 per group): right ventricular systolic pressure (RVSP). f, g) Morphological analysis of pulmonary vessels ( n =5 per group): Data are provided for fully, partially, or nonmuscularized vessels as a percentage of the total vessel count. f: Quantification. g. Representative images. h, i) Immunofluorescence analysis ( n =4 per group) of CD45 + cell accumulation in the lung parenchyma. h: Quantification. i: Representative images. j, k) 3-Nitrotyrosine staining of lung tissue ( n =4 per group). j: Quantification of nitrotyrosine staining. k: Representative images. l, m) Percentage of EdU-positive cells in mouse PCLS treated with 5% CSE in the presence of either MitoQ or DecylTPPLJ ( n =3 per group). l: Quantification of EdU-positive cells. m: Representative images. Statistical analysis was performed using one-way and two-way ANOVA. Data from panels h were log-transformed prior to statistical analysis. The data are presented as the mean ± SEM. n, o) Summary of findings showing the critical role of COX4I2 in CS-induced pulmonary inflammation and emphysema development and the therapeutic potential of MitoQ (n) and COXI2-dependent regulation of neutrophil migration via pericytes (o).

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Functional Assay, Immunofluorescence, Staining, Transformation Assay, Migration