Fig. 4

The inhibitory effect of KMU-11361 on the activation of TAK1/MAPKs/NF-κB/NRLP3 signaling in macrophage-like THP-1 and human RA-FLS cells. A–E Cells were pre-treated with the indicated concentrations of KMU-11361 (0.3, 0.6, and 1 µM) for 1 h and stimulated with LPS (1 µg/mL) for 1 h. A, C Protein expression levels of p-IKKα/β, p-IκBα, p-NF-κB p65, and NF-κB p65 were assessed using western blotting in macrophage-like THP-1 and human RA-FLS cells. B, D Cells were stained with antibodies to NF-κB p65 (green) and DAPI (blue), then captured at ×200 using a fluorescence microscope (scale bar = 100 μm). E Macrophage-like THP-1 cells were stimulated with LPS (1 µg/mL) and/or ATP (1 mM) for 6 h. The protein expression levels of pro-IL-1β, IL-1β, pro-caspase-1, and caspase-1 for the Sup, as well as NLRP3, ASC, pro-caspase-1, and pro-IL-1β for the Lysate, were assessed using western blot analysis. F Immunofluorescence analysis of ASC speck formation in macrophage-like THP-1 cells pretreated with compound KMU-11361 (0.6 µM) for 1 h, followed by stimulation with LPS (1 µg/mL) for 2 h and ATP (2 mM) for 30 min. Cells were stained with an anti-ASC antibody (green) to visualize ASC specks, which are indicated by arrows. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. The expression level of β-actin was used as the protein loading control