Fig. 6

APG-2575 induced NLRP3 transcription by enhancing the nuclear localization of NF-κB. A NF-κB p65 localization in IL-4-activated BMDMs with or without APG-2575 treatment was examined using a confocal fluorescence microscope. Green, NF-κB p65; blue, DAPI. Scale bar, 20 μm. B Luciferase reporter assays with distinct Nlrp3 reporters in Raw264.7 cells activated by IL-4 and then treated with APG-2575 or control. C Luciferase reporter assays with different versions of the 1.61 kb Nlrp3 reporters in RAW264.7 cells activated by IL-4 and then treated with APG-2575 or control. D EMSAs were performed in nuclear extracts with a biotin-labeled NF-κB probe (containing the NF-κB consensus binding sequence). IL-4-activated RAW264.7 cells were treated with APG-2575 or control. E ChIP assay showing the recruitment of NF-κB p65 to the Nlrp3 promoter in IL-4-activated BMDMs. F The simulated complex structure and binding mode of APG-2575 with the RELA protein. G Chemical structures of APG-2575 and biotin-labeled APG-2575 (Bio-APG-2575). H Bio-APG-2575 was added to streptavidin-agarose beads, and the mixture was incubated. Biotin alone was used as a control. Lysates were prepared from BMDMs. I BMDMs were transfected with WT (wild type) NF-κB p65 or mutant NF-κB p65 (Arg33A/Lys56A/Asp277A/Arg278A). Lysates were used for pulldown assays to detect APG-2575 binding using the pulldown assay procedure described in (H). J NF-κB p65 localization in IL-4-activated BMDMs transfected with WT NF-κB p65 or mutant NF-κB p65 and treated with or without APG-2575 was examined using a confocal fluorescence microscope. Green, NF-κB p65; blue, DAPI. Scale bar, 20 μm. K Western blot analysis of NOS2, NLRP3, Arg-1, and NF-κB p65 in IL-4-activated BMDMs transfected with WT NF-κB p65 or mutant NF-κB p65 and treated with or without APG-2575. L Flow cytometric analysis of CD86 and CD206 in IL-4-activated BMDMs transfected with WT NF-κB p65 or mutant NF-κB p65 and treated with or without APG-2575