ly6g gr1  (Bio-Rad)

Journal: Molecular Metabolism

Article Title: Leukocyte-type 12/15-lipoxygenase is essential for timely inflammation-resolution and effective tissue regeneration following skeletal muscle injury

doi: 10.1016/j.molmet.2025.102224

Figure Lengend Snippet: Alox15 −/− mice are deficient in intramuscular 12/15-LOX-derived lipid mediators and display chronic low-grade muscle inflammation. A: Western blot analysis of the abundance of the 12/15-LOX protein in TA muscle homogenates from WT and Alox15 −/− mice. A spleen homogenate from a WT mouse served as a positive control. B: Densitometry quantification of the abundance of the 12/15-LOX protein in the TA muscle. Protein expression was normalized to GAPDH abundance. C: Alox1 5 mRNA expression in the TA muscle as determined by real-time quantitative reverse transcription PCR (RT-qPCR). Gene expression was normalized to Gapdh . D: Heatmap of the top 30 most differentially abundant lipid mediators TA muscle homogenates between WT and Alox15 −/− mice as identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). E-G: Quantification of the intramuscular concentration (pg/mg) of major 12/15-LOX metabolites including 12-hydroxy-eicosatetraenoic acid (12-HETE) ( E ), 15-hydroxy-eicosatetraenoic acid (15-HETE) ( F ), and 14-hydroxy-docosahexaenoic acid (14-HDoHE) ( G ) in TA muscle homogenates as determined by LC-MS/MS. H: TA muscle cross-sections were stained with primary antibodies against the neutrophil marker GR1, the pan monocyte/macrophage marker CD68, or the M2 macrophage marker CD163. Scale bars are 100 μm. I–K: Quantitative analysis of intramuscular numbers of neutrophils (GR1 + cells) ( I ), monocyte/macrophages (CD68 + cells) ( J ), and M2-like macrophages (CD68 + CD163 + cells) ( K ). L-M: Quantification of percentage TA muscle fiber type ( L ) and fiber type specific myofiber cross-sectional area (CSA) ( M ) as determined by MuscleJ 1.0.2 software. Bars show the mean ± SEM of 4–5 mice per group (biological replicates) with dots representing data from each individual mouse. P- values were determined by two-tailed unpaired t-tests. ∗ p < 0.05, ∗∗ p < 0.01 for WT vs. Alox15 −/− mice.

Article Snippet: Primary antibodies used include MyHC type I [Developmental Studies Hybridoma Bank (DSHB), BA-D5c, 1:100], MyHC type IIA (DSHB, SC-71c, 1:100), MyHC type IIB (DSHB, BF–F3c, 1:100), eMHC (DSHB, F1.652s, 1:20), Ly6G (GR1) (Bio-Rad, MCA2387, 1:50), CD68 (Bio-Rad, MCA1957, 1:200), CD163 (Santa Cruz, sc-58965, 1:200), and laminin (Abcam, ab7463, 1:200).

Techniques: Derivative Assay, Western Blot, Positive Control, Expressing, Reverse Transcription, Quantitative RT-PCR, Gene Expression, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Staining, Marker, Software, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Leukocyte-type 12/15-lipoxygenase is essential for timely inflammation-resolution and effective tissue regeneration following skeletal muscle injury

doi: 10.1016/j.molmet.2025.102224

Figure Lengend Snippet: Leukocyte-type 12/15-LOX deficient mice display greater local inflammation and an imbalance of pro-inflammation vs. anti-inflammatory/pro-resolving lipid mediators following acute skeletal muscle injury. A-E: Tibialis anterior (TA) muscle mRNA expression of the pan myeloid cell marker CD11b ( Itgam ) ( A ), the pan monocyte/MФ marker F4/80 ( Adgre1 ) ( B ), the M2 MФ marker CD206 ( Mrc1 ) ( C ), the hematopoietic growth factor M-CSF ( Csf1 ) ( D ), and the hematopoietic growth factor GM-CSF ( Csf2 ) ( E ) in wild type (WT) and Alox15 −/− mice at day 0 (D0), day 3 (D3), day 5 (D5), and day 14 (D14) following myofiber injury induced by intramuscular injection of 50 μL of 1.2% barium chloride (BaCl 2 ). Expression of genes of interest was normalized to Gapdh . F: Immunofluorescence staining with primary antibodies against neutrophils (PMNs) (GR1), monocytes/MФ (CD68), and M2 MФ (CD163) in injured TA muscle of WT and Alox15 −/− mice on D3 and D5 post-injury. Cell nuclei were stained with DAPI and a primary antibody against laminin was used to identify muscle fiber boundaries. Scale bars are 100 μm. G-I : Quantification of PMNs (GR1 + cells/mm 2 ) ( G ), monocytes/MФ (CD68 + cells/mm 2 ) ( H ), and M2 MФ (CD68 + CD163 + cells/mm 2 ) ( I ) at D0, D3, and D5 post-injury in WT and Alox15 −/− mice. J-N: TA mRNA expression of major lipid mediator biosynthesis enzymes including COX-1 ( Ptgs1 ) ( J ), COX-2 ( Ptsg2 ) ( K ), 5-LOX ( Alox5 ) ( L ), 12-LOX ( Alox12 ) ( M ), and 15-LOX ( Alox15 ). O: Heatmap of the top 30 most differentially abundant lipid mediators between WT and Alox15 −/− mice as identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of TA muscle homogenates. P-Q: Quantification of major representative metabolites of the COX pathway (e.g., PGE 2 ) ( P ), 5-LOX pathway (e.g., 5-HETE) ( Q ), 12-LOX pathway (e.g., 14-HDoHE) ( R ), 15-LOX pathway (e.g., 17-HDoHE) ( S ), CYP pathway [e.g., 11(12)-EpETrE) ( T ), and downstream bioactive SPMs (e.g., RvD6) ( Q ). Bars show the mean ± SEM of 4–5 mice per group (biological replicates) with dots representing data from each individual mouse. P- values were determined by two-way ANOVA followed by Holm-Šídák post-hoc tests. ∗ p < 0.05 vs. D0 and # p < 0.05 for WT vs. Alox15 −/− mice.

Article Snippet: Primary antibodies used include MyHC type I [Developmental Studies Hybridoma Bank (DSHB), BA-D5c, 1:100], MyHC type IIA (DSHB, SC-71c, 1:100), MyHC type IIB (DSHB, BF–F3c, 1:100), eMHC (DSHB, F1.652s, 1:20), Ly6G (GR1) (Bio-Rad, MCA2387, 1:50), CD68 (Bio-Rad, MCA1957, 1:200), CD163 (Santa Cruz, sc-58965, 1:200), and laminin (Abcam, ab7463, 1:200).

Techniques: Expressing, Marker, Injection, Immunofluorescence, Staining, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: HemaSphere

Article Title: Inhibiting the alarmin‐driven hematopoiesis‐stromal cell crosstalk in primary myelofibrosis ameliorates bone marrow fibrosis

doi: 10.1002/hem3.70179

Figure Lengend Snippet: S100A9‐inhibition using tasquinimod reveals novel transcriptome reprogramming and reversal of TGFβ activation. (A) Experimental scheme of RNAseq sorting strategy for JAK2 WT or JAK2 V617F CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells, either treated with vehicle or tasquinimod ( n = 3 mice/group/cell population). (B) Principal component analysis of JAK2 WT or JAK2 V617F CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells, either treated with vehicle or tasquinimod. (C) Heatmap representation of PROGENy analysis in CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells. (D) Heatmap representation of extracellular matrix (ECM)‐related Hallmark pathways in CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells, comparing JAK2 V617F vehicle versus JAK2 WT vehicle (labeled as “V617F veh”) and JAK2 V617F tasquinimod versus JAK2 V617F vehicle (labeled as “V617F Tasq”). (E) Overall cell‐to‐cell interactions between CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells. (F) Top 30 deregulated interactions mediated by TGFB1 between CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells. Interactions ordered based on the difference in mean LR expression between JAK2 V617F tasquinimod and JAK2 V617F vehicle.

Article Snippet: The cells were lineage‐depleted using biotinylated antibodies directed against lineages (CD5, CD45R, CD11b, Gr1, 7‐4, and Ter119) (Miltenyi Biotec) and additionally added CD45‐ and CD71‐biotin antibodies (BioLegend).

Techniques: Inhibition, Activation Assay, Labeling, Expressing

Journal: HemaSphere

Article Title: Inhibiting the alarmin‐driven hematopoiesis‐stromal cell crosstalk in primary myelofibrosis ameliorates bone marrow fibrosis

doi: 10.1002/hem3.70179

Figure Lengend Snippet: Tasquinimod treatment abrogates Myc activation and induces JAK2 V617F ‐specific apoptosis. (A) Hallmark gene set enrichment analysis (GSEA) of Gr1 − CD11b + monocytes and (B) Lin − Sca1 + stromal cells, comparing JAK2V617F vehicle versus JAK2WT vehicle (labeled as “V617F vehicle”) and JAK2V617F tasquinimod versus JAK2V617F vehicle (labeled as “V617F tasquinimod”). (C) Frequency of GFP + cells in peripheral blood over time. (D) Flow analysis of early‐stage cell apoptosis (Annexin V + ), and late‐stage apoptosis (Annexin V + 7‐AAD + ) in JAK2 WT ‐HoxB8‐Flt3 or JAK2 V617F ‐HoxB8‐Flt3 cells treated with 50 µM tasquinimod or DMSO control for 24 h. (E) Heatmaps of differential analysis focused on damage‐associated molecular pattern (DAMP)‐associated genes. All comparisons are JAK2 V617F vehicle versus JAK2 WT vehicle (labeled as “V617F vehicle”) and JAK2 V617F tasquinimod versus JAK2 V617F vehicle (labeled as “V617F tasquinimod”). (F) Enzyme‐linked immunosorbent assay (ELISA) analysis of the S100A8/S100A9 heterodimer on murine plasma samples from JAK2 WT or JAK2 V617F animals, either treated with vehicle or tasquinimod drinking water ( n = 7–10/group). (G) Sankey plot showing S100a8‐Tlr4 ligand–receptor interactions in Gr1 − CD11b + monocytes. (H) CARNIVAL‐based pathway inference of the effect of S100a8/S100a9 binding to Tlr4.

Article Snippet: The cells were lineage‐depleted using biotinylated antibodies directed against lineages (CD5, CD45R, CD11b, Gr1, 7‐4, and Ter119) (Miltenyi Biotec) and additionally added CD45‐ and CD71‐biotin antibodies (BioLegend).

Techniques: Activation Assay, Labeling, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Binding Assay