Fig. 3. Septin 11 mediates 27HC activation of endothelial inflammation via ERα.
a–c 27HC activation of NF-κB activity in HAEC via ERα. a, NF-κB luciferase activity was measured in HAEC treated with TNFα (10 ng/ml), E2 (10−8M),TNFα plus E2, or 27HC (20uM) for 6 h (n = 8). b, c Following transduction with control shRNA versus shRNA targeting ERα (b, findings for 3 samples per group are shown), the experiments described in (a) were performed (c, n = 8). d Venn diagrams of proteins identified by LC-MS/MS to have increased or decreased association with ERα upon E2 versus 27HC treatment in HAEC. Diagrams display intersects for common directional changes with the two ligands (upper panel), and opposing directional changes with the two ligands (lower two panels). e Co-immunoprecipitation (IP) of endogenous ERα and septin 11 in HAEC treated with vehicle versus E2 (left panel), or with vehicle versus 27HC (right panel) for 30 min. f HAEC were treated with 27HC, and IP of endogenous ERα was performed and immunoblotting was done for ERα and septin 11 (upper panel), or IP of septin 11 was performed followed by immunoblotting. Septin 11 expression in human atherosclerotic versus control arteries in two independent cohorts. Cohorts I (g) and II (h), which were analyzed by RNAseq and microarray, respectively, contained n = 4 and 201, and 32 and 32 samples respectively. FPKM is fragments per kilobase of transcript per million mapped reads. Box-and-whisker plots are provided in which the central line denotes the median value, the edges represent the upper and lower quartiles, and whiskers indicate the minimum and maximum values. Effect of vehicle versus 27HC on NF-κB activity in HAEC expressing or lacking ERα (i, n = 8) or septin 11 (j, n = 4). In (a), (c), (i) and (j), NF-κB activity is expressed relative to values with vehicle treatment. Data are mean ± SEM. In (a) and (c), P values shown are by Krusakal-Wallis with Dunn’s post hoc testing, and in (g) and (h) two-sided Student’s t tests were used. In (i) and (j), P values by ANOVA with Tukey’s post-hoc testing are shown. The findings in (e) and (f) were confirmed in two independent experiments. Source data are provided as a Source Data file.