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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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Activation of p38^MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. (A,B) p38^MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or SB-202190 (A) or 10 μM PF-3644022 (B) separately, or in combination with SB-202190 (A) or PF-3644022 (B) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3^WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 ^KO cells (Supplementary Fig. S2A,B). (C) Generation of SRC-3^KO A549 cells. Expression of SRC-3 and actin in SRC-3^WT and SRC-3^KO A549 cells were analyzed by Western-blotting. (D) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3^KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3^KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). (E–G) Quantitative presentation of the distribution of SRC-3 in conditions described in (A,B,D) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in (A,B,D) and expressed in percentage. Data in (E,F,G) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. *P < 0.05, **P < 0.01, ***P ≤ 0.001. Now 345.

Activation of p38^MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. (A,B) p38^MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or SB-202190 (A) or 10 μM PF-3644022 (B) separately, or in combination with SB-202190 (A) or PF-3644022 (B) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3^WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 ^KO cells (Supplementary Fig. S2A,B). (C) Generation of SRC-3^KO A549 cells. Expression of SRC-3 and actin in SRC-3^WT and SRC-3^KO A549 cells were analyzed by Western-blotting. (D) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3^KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3^KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). (E–G) Quantitative presentation of the distribution of SRC-3 in conditions described in (A,B,D) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in (A,B,D) and expressed in percentage. Data in (E,F,G) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. *P < 0.05, **P < 0.01, ***P ≤ 0.001. Now 345.