Journal: Immunity

Article Title: Mitochondrial Membrane Potential Regulates Nuclear Gene Expression in Macrophages Exposed to Prostaglandin E2

doi: 10.1016/j.immuni.2018.10.011

Figure Lengend Snippet: (A-B) RNA-seq analysis of gene expression in bone marrow macrophages (BMMs) stimulated for 6 h with: IL-4 (M(IL-4)) or PGE2 (M(−)+PGE2)(A) or M(IL-4) or M(IL-4)+PGE2 (B), compared to no stimulus controls (M(−)) (A,B). (C) Expression of CD301 and RELMα proteins, as measured by flow cytometry, in BMMs stimulated for 24 h with indicated treatments. MFI = mean fluorescence intensity. (D) Western blot of phosphorylated STAT6 (P-STAT6) at indicated times in BMMs treated as indicated. ACTB, actin loading control. (E-F) Extracellular flux analysis (EFA) of oxygen consumption rates (OCR) (E) or extracellular acidification rates (ECAR) (F) at 24 hr post stimulation. For EFA, BMMs were sequentially treated with oligomycin (Oligo), FCCP and rotenone plus antimycin A (R/A) as indicated. (G) 13C-Glucose LC-MS trace into TCA cycle metabolites from cells stimulated for 24 h with indicated treatments. (H-K) ATP concentration (H), cytoplasmic ROS (I), mitochondrial ROS (J), and Δψm as measured by TMRM incorporation (K), in BMMs stimulated as indicated for 24 h. (A-B) Significant (adjusted p value < 0.1) > 2 fold up or down regulated genes based on 3 biological replicates denoted by *. (C) Mean ± SEM from 5 biological replicates (p***<0.0005, p****<0.0001). (G) Mean ± SEM from 3 biological replicates, normalized to M(−) (p**<0.005). (H-K) Mean ± SEM from 5 biological replicates (p*<0.05, p***<0.0005, p****<0.0001). Data are representative of 2 (D) or 3 (C, E, F, H-K) independent experiments. See also Figure S1.

Article Snippet: Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (Qiagen), which makes predictions using pair-wise comparisons between conditions, incorporating in the analysis the statistically significant changes in expression within each of those comparisons. . Flow cytometry Used fluorochrome-conjugate monoclonal antibodies included: CD301 (Milteny Biotech, clone: REA687), CD11b (Biolegend, clone: M1/70), F4/80 (Biozol, clone: BM8), TIM4 (BioLegend, clone: F31–5G3), CD45.1 (BioLegend, clone: A20).

Techniques: RNA Sequencing, Gene Expression, Expressing, Flow Cytometry, Fluorescence, Western Blot, Control, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

Journal: iScience

Article Title: A pref-1-controlled non-inflammatory mechanism of insulin resistance

doi: 10.1016/j.isci.2023.106923

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Article Snippet: CD301–Alexa Fluor 647 , AbD Serotec , Cat# MCA2392A647T; RRID: AB_872014.

Techniques: Recombinant, Protease Inhibitor, Membrane, Western Blot, SYBR Green Assay, cDNA Synthesis, Transfection, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Immunoprecipitation, Solvent, Negative Control, Software, Purification

Journal: Frontiers in Immunology

Article Title: Machine learning identifies PYGM as a macrophage polarization–linked metabolic biomarker in rectal cancer prognosis

doi: 10.3389/fimmu.2025.1639303

Figure Lengend Snippet: PYGM promotes colorectal cancer cell proliferation, migration, invasion, and M2 macrophage polarization. (A) Protein expression levels of PYGM were detected by western blotting. (B) EDU assay analysis. (C) Apoptosis rate was detected by flow cytometry. (D–F) Migration and invasion assay analysis. (G) Schematic illustration of the indirect co-culture system between colorectal cancer cells and THP-1-derived macrophages. (H) The expression levels of CD206 and CD301 were determined by flow cytometry. (I) Arg1, CD206, CD301 and IL-10 gene expression levels were detected by RT-PCR. (J) The protein levels of Arg1, CD206, CD301 and IL-10 were detected by western blotting. *p< 0.05; **p< 0.01; ***p< 0.001, compared to the corresponding groups.

Article Snippet: Western blotting used the following primary antibodies: PYGM (1:1,000; ProteinTech, 19716-1-AP), β-actin (1:4,000; ProteinTech, 66009-1-Ig), Arg1(1:1,000; ProteinTech, 16001-1-AP), CD301(1:1,000; ProteinTech, 13590-1-AP), CD206(1:1,000; ProteinTech, 32647-1-AP), IL-10(1:1,000; ProteinTech, 60269-1-Ig).

Techniques: Migration, Expressing, Western Blot, EdU Assay, Flow Cytometry, Invasion Assay, Co-Culture Assay, Derivative Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Cell

Article Title: A blood atlas of COVID-19 defines hallmarks of disease severity and specificity

doi: 10.1016/j.cell.2022.01.012

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Article Snippet: CD301 (H037G3)-167Er , Fluidigm , Cat# 3167015B, RRID: AB_2892694.

Techniques: Mass Cytometry, Flow Cytometry, Recombinant, Staining, Selection, Antibody Labeling, Labeling, Isolation, Sample Prep, Luminex, Quantitative Proteomics, Generated, Gene Expression, Clone Assay, Marker, Expressing, Mass Spectrometry, Derivative Assay, RNA Sequencing, Sequencing, Illumina Sequencing, Software, Variant Assay