Fig 1: Cinacalcet suppresses the PKCδ/ERK/P65 signaling pathway. (A) Visualization of predicted the binding sites of cinacalcet with respect to NK1R. Structures on the yellow color and grey surface represent the NK1R structure, blue structures represent the amino acids of cinacalcet that bind to NK1R, and structures in pink represent structures of cinacalcet. (B) DARTs assay to test the binding of NK1R and cinacalcet. (C) Representative western blot results to show that cinacalcet suppresses the PKCδ/ERK/P65 signaling pathway. Bone marrow-derived macrophages (BMDMs) were starved with 2% FBS overnight. After treating the cells with cinacalcet (1 μM) for 2 h, TNFα (10 ng/ml) was added for different periods (0, 15, 30, 60 min). Proteins were extracted from the cells, and western blotting was performed. (D) Quantification of p-PKCδ expression. (E) Quantification of p-ERK expression. (F) Quantification of p-P65 expression. (G, H) qRT-PCR was performed to test the mRNA expression levels of IL-1β and IL-6 in BMDMs after stimulation with TNFα (10 ng/ml) in the presence or absence of cinacalcet for 24 h (I, J) ELISA was performed to detect IL-1β and IL-6 levels in BMDM supernatants after stimulation with TNFα (10 ng/ml) in the presence or absence of cinacalcet for 24 h. (K) Proposed model explaining the anti-TNF activity of cinacalcet through direct targeting of the NK1R pathway. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are shown as the mean with standard deviation. Three independent experiments were performed.
Fig 2: PTX3 promotes M2-like macrophage polarization. A, B The mRNA expression of M2 and M1 markers in THP-1 macrophages treated with or without 50 ng/ml PTX3. C The mRNA expression of M2 markers in PTX3-treated THP-1 macrophages with 250 ng/ml IgG1κ or WHC-001 (αPTX3Ab). D The mRNA expression of Arg1, Cd206, and Il1b in bone marrow-derived macrophages treated with or without 100 ng/ml PTX3. E Flow cytometric analysis of M1- and M2-like macrophages in BMDMs co-cultured with MEFs-shVoid, -shPtx3#915,or shPtx3#916 for 48 h. The CD45 + CD11b + F4/80 + cells were characterized by M1-like (CD86 + CD206−) and M2-like (CD206 + CD86−) macrophages through flow cytometric analysis. F The mRNA expression of Ptx3, Arg1, and Cd206 in tamoxifen-induced Ptx3fl/fl or Ptx3fl/fl; Ubc-Cre bone marrow-derived macrophages (M-CSF-primed M0 BMDMs) following stimulation with 20 ng/ml mIL-13 and 20 ng/ml mIL-4 (M2 polarization). G The mRNA expression of IFNG and TNFA in activated Jurkat cells (stimulated with PMA + ionomycin) incubated with conditioned medium from untreated THP-1 macrophages (Ctrl-CM) or PTX3-treated THP-1 macrophages (PTX3-CM). H Angiogenesis of HUVECs incubated in serum-free medium (No cell-CM), conditioned-medium from untreated THP-1 macrophages (Ctrl-CM) or conditioned medium from PTX3-treated THP-1 macrophages (PTX3-CM). The relative angiogenic ability was determined by the number of branch sites/nodes of tubes per field of view. I Western blot analysis of recombinant wild-type and N220A mutant PTX3 proteins treated with or without PNGase. J The mRNA expression of M2 markers in THP-1 macrophages treated with or without 50 ng/ml recombinant N220A mutant PTX3 protein. p values were calculated by two-tailed paired Student’s t test, one-way ANOVA, or two-way ANOVA. ns represents nonsignificant difference,*represents a p value of < 0.05, **represents a p value of < 0.01, and ***represents a p value of < 0.001. Values on the plots are presented as the means ± SEMs. Data are combined from 3 to 5 independent experiments
Fig 3: Comparison of Inflammatory Cytokine Concentrations Across Treatment Groups in Mice(A: IFN-γ, B:IL-17A, C:TNF-ɑ)
Fig 4: Regional differences in cell alignment and monocyte adhesion in 3D‐printed carotid artery constructs. A) Immunofluorescence of hAECs (CD31, green) and hASMCs (α‐SMA, purple) in the CCA (zone 1) and carotid bifurcation (zone 2) after 3 days of static and 8 days of perfusion culture. Nuclei stained with DAPI (blue). B) Schematic showing flow direction and cell nuclei orientation; cell alignment is defined as 45°–90° to the horizontal. C) Quantification of % hAECs aligned to flow in each zone. D,E) Polar plots showing endothelial alignment across four angular bins. F) Monocyte (U937, blue) adhesion on inflamed hAECs (red, Phalloidin) in zones 1 and 2. G) Quantification of monocyte adhesion post‐TNF stimulation. Data shown as mean ± SD (n = 4); **** p < 0.0001, Student's t‐test.
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