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. 2019 Feb 28;216(4):807–830. doi: 10.1084/jem.20171438

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© 2019 Mentrup et al.

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Figure 7. — Accumulation of the LOX-1 NTF induces a pro-atherogenic state in endothelial cells. (A) Expression of ICAM-1 was analyzed in control (–) or LOX-1 NTF transduced iMAECs by Western blotting. (B) Quantification of A. N = 2, n = 6. Student’s t test. (C) Up-regulation of surface ICAM-1 in LOX-1 NTF transduced iMAECs was validated by flow cytometry. As a control, cells were treated with 5 ng/ml TNF. N = 2, n = 6. One-way ANOVA with Tukey’s post hoc test. (D) Up-regulation of Icam-1 was validated by qPCR. N = 2, n = 6. Student’s t test. (E) Candidate genes from endothelial cell biology and atherosclerosis RT² Profiler arrays with differential regulation between control and iMAEC NTF cells. (F) Differences in mRNA levels of the selected genes were validated by qPCR. N = 2–3, n = 6–9. Student’s t test. (G) Secretion of CTGF was blocked in iMAEC control (−) or NTF cells by incubation with Brefeldin A (1 µg/ml) for 6 h. Intracellular CTGF levels were analyzed by Western blotting. (H) Quantification of G. N = 2, n = 12. Student’s t test. (I–L) iMAEC control or NTF cells were treated for 3 h with 25 µM U0126 (MEK-Inh.), 1 µM Saracatinib (Src-Inh.), or 10 µM Y-27632 (ROCK-Inh.) or left untreated as indicated. Icam-1 (I, N = 3–4, n = 9–12), Ctgf (K, N = 3, n = 8–9), and Pdgfb (L, N = 3, n = 8–9) mRNA levels were quantified by qPCR. One-way ANOVA with Dunnett’s post hoc test. (M) Up-regulation of validated candidate genes was monitored in iMAECs treated for 16 h with either DMSO or 40 µM ZLL. N = 2–3, n = 6–9. Student’s t test. (N) Up-regulation of ICAM-1 upon ZLL administration was validated by flow cytometry. N = 3, n = 11. Student’s t test. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant. N, the number of independent experiments; n, the number of individual samples for quantification. All data are shown as mean ± SD.