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. 2020 Jul 9;44(3):1049–1063. doi: 10.3892/or.2020.7683

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Copyright: © Pan et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Figure 5. — Effects of shikonin on IL-6-induced STAT3 activation in A549 and H1299 cells. (A) Expression levels of phosphorylated (p)-STAT3 and STAT3 were detected by western blotting after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. Protein expression levels were semi-quantified by densitometric analysis. *P<0.05, **P<0.01, compared with the IL-6-treated group. (B) Immunofluorescence assays were performed to determine the effects of shikonin on the nuclear translocation of p-STAT3 after treatment with shikonin (1.25 µM) and IL-6 (50 ng/ml) for 24 h. Upper panels, representative immunofluorescence images of p-STAT3 staining (magnification ×800). Lower panel, the percentage of cells with p-STAT3 nuclear translocation. **P<0.01, compared with the IL-6-treated group. (C) Transactivation activities of p-STAT3 in A549 and H1299 cells co-transfected with pSTAT3-TA-luc and pRL-TK Renilla, and treated with shikonin and IL-6 (50 ng/ml) for 24 h. Luciferase activity was detected by the Dual Luciferase Reporter kit and normalized against the values for the corresponding pRL-TK Renilla activity. *P<0.05, **P<0.01, compared with the IL-6-treated group. (D) The protein expression levels of p-JAK2 and JAK2 were detected by western blotting after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. β-actin was used as an internal control. Protein expression levels were semi-quantified by densitometry analysis. (E and F) After transfection with STAT3 plasmids for 24 h, the cells were treated with shikonin (1.25 µM) and IL-6 (50 ng/ml) for another 24 h. (E) Expression levels of p-STAT3, Flag-STAT3, and STAT3 proteins were detected by western blotting. (F) mRNA expression levels of epithelial-mesenchymal transition (EMT)-related genes (E-cadherin, N-cadherin, vimentin and Snail) were detected by real-time PCR. Data are shown as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, compared with the IL-6-treated group; ^#P<0.05, ^##P<0.01, compared with the combined shikonin (1.25 µM) and IL-6 (50 ng/ml) treatment group. IL-6, interleukin-6; STAT3, signal transducer and activator of transcription 3; JAK, Janus kinase.

Figure 5. — Effects of shikonin on IL-6-induced STAT3 activation in A549 and H1299 cells. (A) Expression levels of phosphorylated (p)-STAT3 and STAT3 were detected by western blotting after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. Protein expression levels were semi-quantified by densitometric analysis. *P<0.05, **P<0.01, compared with the IL-6-treated group. (B) Immunofluorescence assays were performed to determine the effects of shikonin on the nuclear translocation of p-STAT3 after treatment with shikonin (1.25 µM) and IL-6 (50 ng/ml) for 24 h. Upper panels, representative immunofluorescence images of p-STAT3 staining (magnification ×800). Lower panel, the percentage of cells with p-STAT3 nuclear translocation. **P<0.01, compared with the IL-6-treated group. (C) Transactivation activities of p-STAT3 in A549 and H1299 cells co-transfected with pSTAT3-TA-luc and pRL-TK Renilla, and treated with shikonin and IL-6 (50 ng/ml) for 24 h. Luciferase activity was detected by the Dual Luciferase Reporter kit and normalized against the values for the corresponding pRL-TK Renilla activity. *P<0.05, **P<0.01, compared with the IL-6-treated group. (D) The protein expression levels of p-JAK2 and JAK2 were detected by western blotting after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. β-actin was used as an internal control. Protein expression levels were semi-quantified by densitometry analysis. (E and F) After transfection with STAT3 plasmids for 24 h, the cells were treated with shikonin (1.25 µM) and IL-6 (50 ng/ml) for another 24 h. (E) Expression levels of p-STAT3, Flag-STAT3, and STAT3 proteins were detected by western blotting. (F) mRNA expression levels of epithelial-mesenchymal transition (EMT)-related genes (E-cadherin, N-cadherin, vimentin and Snail) were detected by real-time PCR. Data are shown as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, compared with the IL-6-treated group; ^#P<0.05, ^##P<0.01, compared with the combined shikonin (1.25 µM) and IL-6 (50 ng/ml) treatment group. IL-6, interleukin-6; STAT3, signal transducer and activator of transcription 3; JAK, Janus kinase.