Fig 1: Human atherosclerotic plaques exhibit a significant upregulation of p300, HAT1, H3K27ac, and Nox5 proteins. Note the densitometric analysis of type A-HAT (p300) (a), type B-HAT (HAT1) (b), H3K27ac (e), and Nox5 (f) protein levels analyzed by western blot of atherosclerotic (carotid artery) and nonatherosclerotic (superior thyroid artery) tissues derived from patients undergoing extended endarterectomy. n = 8‐11; ∗∗P < 0.01, ∗∗∗P < 0.001. P values were taken in relation to nonatherosclerotic condition. Representative immunoblots depicting the induction of p300 (c), HAT1 (d), H3K27ac (g), and Nox5 (h) proteins in atherosclerotic carotid arteries. Non-athero: nonatherosclerotic tissue; Athero: atherosclerotic tissue.
Fig 2: (A) Effect of 50 μM NS1 (mixture of isomers), NS1-2, NS1-3 on the migration of SUM159 breast cancer cells, see also Supplementary Figure S10; (B) Western blot analysis of NOX2 in SUM159 cells treated or not with NS1 (50 µM) and their quantification after 48 h treatment; (C,D) Representative Western blots of NOX2, NOX1, NOX3 and NOX5 in SUM159 cells treated or not with NS1 or its isomers for 48 h and their quantification.
Fig 3: Exposure of cultured human Mac to LPS induces the upregulation of HAT1, H3K27ac, H3K9ac, and Nox5 protein levels in a concentration-dependent manner. Note the densitometric analysis of HAT1 (a), H3K27ac (b), H3K9ac (e), and Nox5 (f) protein levels analyzed by western blot in Mac exposed to increasing concentrations of LPS (0.1-1 μg/mL). n = 3‐4; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. P values were taken in relation to vehicle-treated cells. Representative immunoblots depicting the gradual upregulation of HAT1 (c), H3K27ac (d), H3K9ac (g), and Nox5 proteins (h) in LPS-challenged cells.
Fig 4: IHC localization of p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins in the area of infiltrated immune cells/Mac, fibrous cap, and lipid-rich core of a human carotid atherosclerotic plaque. The images ((a) 4x magnification, (b) 10x magnification) show representative IHC staining of 5 μm thick serial sections from a human carotid atherosclerotic plaque for p300, HAT1, H3K27ac, H3K9ac, Nox5, CD45, CD68, and αSMA proteins. The red lesional lipid deposits were detected by Oil Red O staining. The positively stained cells are marked with arrows. Note the localization of Nox5 protein in the area of fibrous cap and lipid-rich core of atherosclerotic plaque expressing markers of infiltrated immune cells (CD45, CD68) and vascular SMCs (αSMA). The images are representative of 4 independent IHC assays.
Fig 5: Overexpression of p300 or HAT1 upregulates the promoter activity of human Nox5 gene in Mac. The RAW264.7 cells were transiently transfected with Nox5 gene promoter-luciferase reporter gene constructs in the presence of empty vector or p300/HAT1 expression vectors. Representative agarose gel electrophoresis showing the digestion products of the c1-c8 constructs (a). Schematic depiction of the 5′-deletion mutants of the Nox5 gene promoter used in the cotransfection assays (b). Induction of luciferase level directed by the DNA regulatory elements derived from human Nox5 gene promoter in response to p300 or HAT1 overexpression in cultured Mac (c). n = 4; ∗∗∗P < 0.001. P values were taken in relation to the corresponding empty vector control.
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