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. 2019 Mar 28;23:101175. doi: 10.1016/j.redox.2019.101175

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© 2019 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 2 — Effects of 15-keto PGE₂on phosphorylation, dimerization, nuclear localization, and transcriptional activities of STAT3 in MCF10A-ras cells. A. MCF10A-ras cells were treated with indicated concentrations of 15-keto PGE₂ for 24 h. The expression levels of STAT3 and P-STAT3^Y705 were determined by Western blot analysis. **p < 0.01. B. MCF10A-ras cells were incubated with 15-keto PGE₂ (20 μM) for indicated time periods. The protein level of phosphorylated STAT3 was measured by Western blotting. C. Whole lysates from MCF10A-ras cells treated with or without 15-keto PGE₂ (20 μM) for 12 h were prepared for immunoprecipitation of STAT3 protein using protein A/G agarose conjugated C-terminus anti-STAT3 antibody. The STAT3 homo-dimerization was analyzed by Western blot analysis using N-terminus anti-STAT3 antibody. ***p < 0.001. D. MCF10A-ras cells were treated with 15-keto PGE₂ (20 μM) for 24 h, and nuclear extracts were immunoblotted for detection of P-STAT3^Y705. Lamin B was used as a nuclear protein marker. **p < 0.01. E. Immunocytochemical analysis was performed using antibodies against P-STAT3^Y705. MCF10A-ras cells were treated with or without 15-keto PGE₂ (20 μM) for 24 h. Nuclei were stained with DAPI and visualized under a confocal microscope. Two images were merged to verify co-localization. F. The luciferase assay was performed with HeLa/P-STAT3-luc reporter cells. The cells were treated with 15-keto PGE₂ (10 or 20 μM) for 24 h and then stimulated with OSM (10 ng/ml) for another 6 h. Cells were then analyzed by a microplate luminometer. **p < 0.01, ***p < 0.001.

Fig. 2 — Effects of 15-keto PGE₂on phosphorylation, dimerization, nuclear localization, and transcriptional activities of STAT3 in MCF10A-ras cells. A. MCF10A-ras cells were treated with indicated concentrations of 15-keto PGE₂ for 24 h. The expression levels of STAT3 and P-STAT3^Y705 were determined by Western blot analysis. **p < 0.01. B. MCF10A-ras cells were incubated with 15-keto PGE₂ (20 μM) for indicated time periods. The protein level of phosphorylated STAT3 was measured by Western blotting. C. Whole lysates from MCF10A-ras cells treated with or without 15-keto PGE₂ (20 μM) for 12 h were prepared for immunoprecipitation of STAT3 protein using protein A/G agarose conjugated C-terminus anti-STAT3 antibody. The STAT3 homo-dimerization was analyzed by Western blot analysis using N-terminus anti-STAT3 antibody. ***p < 0.001. D. MCF10A-ras cells were treated with 15-keto PGE₂ (20 μM) for 24 h, and nuclear extracts were immunoblotted for detection of P-STAT3^Y705. Lamin B was used as a nuclear protein marker. **p < 0.01. E. Immunocytochemical analysis was performed using antibodies against P-STAT3^Y705. MCF10A-ras cells were treated with or without 15-keto PGE₂ (20 μM) for 24 h. Nuclei were stained with DAPI and visualized under a confocal microscope. Two images were merged to verify co-localization. F. The luciferase assay was performed with HeLa/P-STAT3-luc reporter cells. The cells were treated with 15-keto PGE₂ (10 or 20 μM) for 24 h and then stimulated with OSM (10 ng/ml) for another 6 h. Cells were then analyzed by a microplate luminometer. **p < 0.01, ***p < 0.001.