Fig. 5

TNF-α increases LOX-1 expression at the cellular plasma membrane by a mechanism mediated by the TNF-α receptor activity and the ROS/NOX pathway and NF-κB activation. Cells were exposed to TNF-α and LOX-1 expression was analyzed. a Representative images from western blot experiments performed for detection of LOX-1 in the presence of TNF-α (0, 5, and 10 ng/ml) for 24 h. b Densitometric analyses of the experiments shown in a. Protein levels were normalized against tubulin, and data are expressed normalized to the TNF-α 0 ng/ml condition (N = 4). Statistical differences were assessed by a one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test. *P < 0.05. **P < 0.01. Graph bars show the mean ± SD. c and d LOX-1 expression in plasma membrane-rich and cytosol-rich fractions from ECs exposed to TNF-α (10 ng/ml) for 24 h. Protein levels were normalized against Na⁺ pump in the membrane-rich fraction. Data are expressed normalized to vehicle condition (N = 3). e and f LOX-1 expression in the plasma membrane-rich fraction from ECs exposed to TNF-α (10 ng/ml) for 24 h in the absence (–) or presence (+) of the TNF-α receptor inhibitor, R7050 (5 μM) (N = 3). Data are expressed normalized to vehicle condition. Statistical differences were assessed by a two-way analysis of variance (ANOVA) followed by Tukey post hoc test. **P < 0.01. Graph bars show the mean ± SD. TNF-α-induced NOX isoform mRNA expression was determined by RT-qPCR in HAEC (g) and HUVEC (h). Data are expressed normalized to vehicle condition (N = 3). Statistical differences for HAEC and HUVEC samples were assessed by a one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test. *P < 0.05. **P < 0.01. Graph bars show the mean ± SD. LOX-1 expression was measured in the plasma membrane-rich fraction from cells exposed to TNF-α 10 ng/ml for 24 h in the absence (–) or presence (+) of NAC (5 mM) (i, j), GSH (1 mM) (k, l), the NAD(P)H oxidase inhibitor DPI (10  μM) (m and n), NAD(P)H oxidase inhibitor Apocynin (10 mM) (o and p) and the NF-κB inhibitor SC3060 (5 μM) (q and r) for 24 h (N = 3). Statistical differences were assessed by a two-way analysis of variance (ANOVA) followed by Tukey post hoc test. **P < 0.01. Graph bars show the mean ± SD