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. 2020 Jul 9;10:11388. doi: 10.1038/s41598-020-68219-4

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© The Author(s) 2020

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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SRC-3 is required for MK2-mediated induction of IL-6 expression in response to TNF-α. (A,B) SRC-3 is involved in NF-κB activation. (A) SRC-3^KO A549 cells were co-transfected with 120 ng κB-ConA-luc vector and 50 ng of either pSG5 empty vector, SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG vector. After 48 h TNF-α was added (if not other indicated 10 ng/ml for 5 h) before determination of luciferase activity relative to pSG5. (B) A549-NF-κB-Luc cells were transfected with scrambled siRNA or SRC-3 siRNA and 48 h later stimulated with TNF-α or left unstimulated. Nontransfected cells were pretreated with PF-3644022 for 30 min before TNF-α treatment. Luciferase activities are shown relative to unstimulated scrambled siRNA. (C,D) SRC-3 is involved in TNF-α-induced IL-6 expression. SRC-3^WT and SRC-3^KO A549 cells were stimulated with TNF-α for 2 h or left unstimulated. mRNA expression of IL-6 (C) and MMP9 (D) were determined relative to GAPDH and TFRC. Fold changes are presented relative to unstimulated SRC-3^WT cells. (E,J) MK2 and p38^MAPK activity are required for transcription of IL-6. A549 cells were transfected with 120 ng pGL3-IL-6-promoter vector and after 48 h treated for 30 min with 0.2 μl DMSO, 10 μM PF-3644022 (E) or SB-202190 (J) followed by stimulation with TNF-α. Luciferase activities are shown relative to DMSO. (F–H) MK2 is involved in TNF-α induced TRAF1 (F), IL-8 (G) and ICAM1 (H) mRNA expression. A549 cells pretreated with DMSO or 10 μM PF-3644022 for 30 min were stimulated with TNF-α for 2 h or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. (I) p38^MAPK is involved in NF-κB-dependent luciferase activity. A549-NF-κB-Luc cells were pretreated with DMSO or SB-202190 and then stimulated with TNF-α or left unstimulated. Luciferase activities are shown relative to DMSO. (K–M) p38^MAPK is involved in TNF-α-induced IL-6 (K) and IL-8 (L) but not MMP9 (M) mRNA expression. A549 cells pretreated with DMSO or 10 μM SB-202190 for 30 min were stimulated with TNF-α or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. Data are presented as mean ± SD (n = 3). Unpaired t-test; *P < 0.05, **P < 0.01, ***P ≤ 0.001.

SRC-3 is required for MK2-mediated induction of IL-6 expression in response to TNF-α. (A,B) SRC-3 is involved in NF-κB activation. (A) SRC-3^KO A549 cells were co-transfected with 120 ng κB-ConA-luc vector and 50 ng of either pSG5 empty vector, SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG vector. After 48 h TNF-α was added (if not other indicated 10 ng/ml for 5 h) before determination of luciferase activity relative to pSG5. (B) A549-NF-κB-Luc cells were transfected with scrambled siRNA or SRC-3 siRNA and 48 h later stimulated with TNF-α or left unstimulated. Nontransfected cells were pretreated with PF-3644022 for 30 min before TNF-α treatment. Luciferase activities are shown relative to unstimulated scrambled siRNA. (C,D) SRC-3 is involved in TNF-α-induced IL-6 expression. SRC-3^WT and SRC-3^KO A549 cells were stimulated with TNF-α for 2 h or left unstimulated. mRNA expression of IL-6 (C) and MMP9 (D) were determined relative to GAPDH and TFRC. Fold changes are presented relative to unstimulated SRC-3^WT cells. (E,J) MK2 and p38^MAPK activity are required for transcription of IL-6. A549 cells were transfected with 120 ng pGL3-IL-6-promoter vector and after 48 h treated for 30 min with 0.2 μl DMSO, 10 μM PF-3644022 (E) or SB-202190 (J) followed by stimulation with TNF-α. Luciferase activities are shown relative to DMSO. (F–H) MK2 is involved in TNF-α induced TRAF1 (F), IL-8 (G) and ICAM1 (H) mRNA expression. A549 cells pretreated with DMSO or 10 μM PF-3644022 for 30 min were stimulated with TNF-α for 2 h or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. (I) p38^MAPK is involved in NF-κB-dependent luciferase activity. A549-NF-κB-Luc cells were pretreated with DMSO or SB-202190 and then stimulated with TNF-α or left unstimulated. Luciferase activities are shown relative to DMSO. (K–M) p38^MAPK is involved in TNF-α-induced IL-6 (K) and IL-8 (L) but not MMP9 (M) mRNA expression. A549 cells pretreated with DMSO or 10 μM SB-202190 for 30 min were stimulated with TNF-α or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. Data are presented as mean ± SD (n = 3). Unpaired t-test; *P < 0.05, **P < 0.01, ***P ≤ 0.001.