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. 2016 Jun 15;9:61–76. doi: 10.1016/j.ebiom.2016.06.017

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© 2016 The Authors

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

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Fig. 5 — Api regulates macrophage polarization via inhibiting the interaction between p65 and PPARγ. (a)The expression of p65 in the primary peritoneal macrophages of 19 weeks ND and HFD mice treated with 30 mg/kg Api for 21 days was assayed by western blotting for three times, 3 mice/time (top). And the quantification of the protein level by using image J software (bottom). All values are expressed as mean ± SEM. Statistical analysis is based on the Student's t-test. *P < 0.05 compared with ND or vehicle. (b)The impact of Api on IκBα, p-IκBα abundance were evaluated in the primary peritoneal macrophages of mice treated for 21 days with 30 mg/kg Api by western blotting for three times, 3 mice/group (top). And the quantification of the protein level by image J software (bottom). All values are expressed as mean ± SEM. Statistical analysis is based on the Student's t-test. **P < 0.01 compared with vehicle group. (c) The effect of 7.5 μM Api in LPS-induced ANA-1 on the activities of p65 was detected by EMSA assay. Figure shows one image from at least three independent experiments. (d) Using immunofluorescence, the p65 was evaluated in macrophage treated with Control, LPS (500 ng/mL), or LPS (500 ng/mL) and 7.5 μM Api simultaneously for 24 h. p65 is shown in red, and nuclei stained with DAPI. Original magnification is × 400. Figure shows one image from at least three independent experiments. (e) The nuclei/cytoplasm ratio of (d) at least three independent experiments was quantified by Image J software. All values are expressed as mean ± SEM. Statistical analysis is based on one-way ANOVA followed by a Dunnett's test. ***P < 0.001 compared with con or LPS group. (f) The p65/PPARγ complex in the ATM of mice treated with 30 mg/kg Api for 21 days was assayed by confocal microscopy. p65 is shown in red, PPARγ is shown in green and nuclei stained with DAPI. Original magnification is × 400. (g) Data from (F) at least three independent experiments was quantified by Image J software. All values are expressed as mean ± SEM. Statistical analysis is based on one-way ANOVA followed by a Dunnett's test. ***P < 0.001 compared with ND or HFD. (h) ANA-1 macrophages were treated with 500 ng/mL LPS, 10 ng/mL IL-4, 500 ng/mL LPS plus 7.5 μM Api simultaneously or 10 ng/mL IL-4 plus 7.5 μM Api for 24 h. The nuclei and cytoplasm protein was lysed and subjected to immunoprecipitation and western blotting (left). Data from at least three independent experiments was quantified by Image J software (right). All values are expressed as mean ± SEM. Statistical analysis is based on one-way ANOVA followed by a Dunnett's test. *P < 0.05, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)