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. 2019 Oct 8;10:4579. doi: 10.1038/s41467-019-12163-z

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© The Author(s) 2019

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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Fig. 5 — ACF re-activated Leflunomide-AHR-CRP signaling in vitro. a Binding of ARNT with HIF1α and level of VEGF in IL-6-pretreated THLE-2 cells incubated with vehicle (DMSO) or ACF (5.0 μM). Pretreatment of IL-6 induced the overexpression of CRP in THLE-2 cells. THLE-2 cells incubated in hypoxia were used as a positive control. b Binding of ARNT with AHR in IL-6-pretreated THLE-2 cells incubated with vehicle (DMSO), Leflunomide (LEF, 10 μM), ACF (5.0 μM) and the combination of Leflunomide and ACF (LEF + ACF, LEF, 10 μM; ACF, 5.0 μM), respectively. c Relative CRP mRNA and CRP release in IL-6-pretreated THLE-2 cells after the treatment. d The diagram of the experimental design. Briefly, THLE-2 cells were pretreated with IL-6 and then incubated with vehicle (DMSO), Leflunomide (LEF, 10 μM), ACF (5.0 μM) and the combination of Leflunomide and ACF (LEF + ACF, LEF, 10 μM; ACF, 5.0 μM), respectively. After 8 h, the medium was replaced, and the THLE-2 cells were continuously cultured for another 48 h. The conditioned mediums were collected for culturing monocytes in the presence of macrophage colony stimulating factor (M-CSF). e TRAP staining (upper) and pit formation assay (bottom) in monocytes incubated with the conditioned mediums. Scale bar, 100 μm. f Quantitative determination of TRAP-positive (TRAP+) multinuclear cells per well and percentage of pit area from pit formation assay. g Relative TRAP5b mRNA level in monocytes incubated with the conditioned mediums. *P < 0.05 as determined by one-way ANOVA with a post hoc test. Each experiment was repeated three times. Source data are provided as a Source Data file