Fig 1: 15-Epi-LXA4 and MaR1 induce SPM synthetic enzymes and ALX in Achilles tendon stromal cells. Tendon stromal cells were derived from patients with AT (n = 5 donors) or AR (n = 5 donors). Cells were incubated with 15-epi-LXA4 (10 nM) or vehicle for 24 h at 37°C and then with IL-1β (10 ng/ml) for 24 h. A) Incubation in 15-epi-LXA4 significantly induced ALOX15 mRNA in both AT (P = 0.01) and AR (P = 0.03) cells compared with respective vehicle controls. Gene expression is normalized to β-actin; bars show median values. B) Representative images of immunocytochemistry for the proresolving receptor ALX (green) and SPM synthetic enzymes ALOX12 (violet) and ALOX15 (red) in IL-1β–stimulated AT and AR tendon stromal cells incubated in 10 nM 15-epi-LXA4, 10 nM MaR1, or vehicle control for 24 h. Cyan represents Popo-1 nuclear counterstain. All images are representative of n = 3 donors. Scale bar, 20 μm.
Fig 2: Lipoxin B4 (LXB4) and resolvin E1 (RvE1) induce specialized proresolving mediator (SPM) biosynthetic enzymes and regulate the proresolving receptor for chemerin and resolvin E1 (ChemR23)/human resolvin E1 (ERV1) in tendon stromal cells. Tendon stromal cells were derived from patients with shoulder tendon tears (TD; n = 6) or healthy volunteers (HV; n = 6). Cells were incubated with 10 nmol/L LXB4, 10 nmol/L RvE1, or vehicle for 24 hours at 37°C then with 10 ng mL-1 of IL-1ß for 24 hours. A: Incubation in LXB4 significantly induces ALOX15 mRNA in both TD and HV cells compared with respective vehicle controls. Incubation in RvE1 significantly induces ALOX15 mRNA in both TD and HV cells compared with respective vehicle controls. Gene expression is normalized to ß-actin. Representative images of immunocytochemistry for the SPM biosynthetic enzymes 15-lipoxygenase (ALOX15) (green) and 12-lipoxygenase (ALOX12) (violet) in IL-1ß–stimulated HV (B) and TD tendon stromal cells (C) incubated in 10 nmol/L LXB4, 10 nmol/L RvE1, or vehicle control for 24 hours. Cyan represents POPO-1 nuclear counterstain. D: IL-1ß–stimulated TD cells show increased ERV1 mRNA expression compared with respective HV cells. Gene expression is normalized to ß-actin. E: Representative images of immunocytochemistry for proresolving receptors lipoxin A4 receptor (ALX) (green), human resolvin E1 (ERV1) (red), and leukotriene B4 receptor (BLT1) (violet) in IL-1ß–stimulated diseased tendon stromal cells incubated in 10 nmol/L LXB4, 10 nmol/L RvE1, or vehicle control for 24 hours. Cyan represents POPO-1 nuclear counterstain. Bars indicate median values. n = 3 donors. Scale bars = 20 µm (B, C, and E). *P < 0.05, **P < 0.01.
Fig 3: Differential regulation of specialized proresolving mediator (SPM) biosynthetic enzyme activity by resolvin E1 (RvE1) and lipoxin B4 (LXB4) in patient-derived and healthy (H) volunteer tendon stromal cells. Tendon stromal cells were derived from the H group (n = 5 donors) and patients with shoulder tendon tears or the diseased (D) group (n = 5 donors). Cells were incubated with 10 nmol/L RvE1, 10 nmol/L LXB4, or vehicle for 24 hours at 37°C then with 10 ng mL−1 of IL-1β for 24 hours. Monohydroxy fatty acids–produced SPM biosynthetic enzymes were identified and quantified using liquid chromatography with tandem mass spectrometry–based lipid mediator profiling. Cumulative concentrations of the 5-lipoxygenase (ALOX5) products 7-hydroxydocosahexaenoic acid, 7-hydroxydocosapentaenoic acid, 5-hydroxyeicosapentaenoic acid, and 5-hydroxyeicosatetraenoic acid (A); the 12-lipoxygenase (ALOX12) products 14-hydroxydocosahexaenoic acid, 14-hydroxydocosapentaenoic acid, 12-hydroxyeicosapentaenoic acid, and 12-hydroxyeicosatetraenoic acid (B); the 15-lipoxygenase (ALOX15) products 17-hydroxydocosahexaenoic acid, 17-hydroxydocosapentaenoic acid, 15-hydroxyeicosapentaenoic acid, and 15-hydroxyeicosatetraenoic acid (C); and cyclooxygenase (COX) products 17-hydroxydocosahexaenoic acid and 17-hydroxydocosapentaenoic acid (D). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01 versus respective vehicle control (Friedman's test followed by Dunn's post hoc test).
Fig 4: Expression of the ALOX gene and protein in ρ0 cells. A, Expression of the ALOX5 gene. B, Expression of the ALOX12 gene. C, Expression of the ALOX15 gene. The results are expressed as the mean ± SEM. **P < .01 using Student's t test. D, Western blotting of ALOX5, ALOX12, and ALOX15. Unlike ALOX5, the expression of ALOX12 and ALOX15 was upregulated in HeLa and SAS ρ0 cells
Fig 5: Biosynthesis of specialized proresolving mediators in tendon stromal cells. A: mRNA expression of enzymes implicated in the biosynthesis of specialized proresolving mediator (SPMs; ALOX15, ALOX12), inflammation-initiating eicosanoids (PTGS2), and the degradation of SPMs and inflammation-initiating eicosanoids (HPGD) in tendon stromal cells from patients with shoulder tendon tears and healthy volunteers (HVs). Incubation in lipoxin B4 (LXB4) significantly induces ALOX15 mRNA in both tendon disease (TD) and HV cells compared with respective vehicle controls. Incubation in resolvin E1 (RvE1) significantly induces ALOX15 mRNA in both TD and HV cells compared with respective vehicle controls. Gene expression is normalized to β-actin. B: Summary of lipid mediator biosynthetic pathways. Bars indicate median values. *P < 0.05 versus respective vehicle control. PD, protectin; RvT, 13-series resolvin; MaR, maresin; RvE, E-series resolvins; LX, lipoxins; LT, leukotrienes; PG, prostaglandins; Tx, thromboxane; ALOX, lipoxygenase; COX, cyclooxygenase; CYP, cytochrome P450.
Supplier Page from Abcam for Anti-12 Lipoxygenase/ALOX12 antibody - C-terminal