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. 2017 Jun 8;7:3057. doi: 10.1038/s41598-017-03130-z

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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TNF-α markedly upregulates CX3CL1 production by HUVECs. (A) HUVECs were stimulated with TNF-α (5 ng/ml) up to 8 hours and CX3CL1 mRNA expression was analyzed by real-time RT-PCR at the indicated time-points. (B) HUVECs were treated with various concentrations of TNF-α (1 to 10 ng/ml) for 4 hours, and the expression of CX3CL1 was analyzed by real-time RT-PCR. (C) HUVECs were stimulated with various concentrations of TNF-α (1 to 10 ng/ml) for 18 hours and the amount of CX3CL1 in the culture supernatant was quantified by conventional ELISA. (D) Purified monocytes were treated with or without IS for 48 hr, and the supernatant (MCM: monocyte-conditioned media) of each culture was harvested. Control or IS-treated monocyte-conditioned media (Con- or IS-MCM) was added to confluent, cultured HUVECs in the presence of anti-TNF-α Ab or control IgG, followed by a 4 hr incubation. CX3CL1 mRNA expression in treated HUVECs was analyzed by real-time RT-PCR. Expression of β-actin was used as a normalization control. Bar graphs show the mean ± SEM of three to four independent experiments. *p < 0.05 and **p < 0.01: compared to no TNF-α treatment group by two-tailed paired t-test (B and C).