Journal: Journal of Pharmaceutical Analysis

Article Title: New applications of clioquinol in the treatment of inflammation disease by directly targeting arginine 335 of NLRP3

doi: 10.1016/j.jpha.2024.101069

Figure Lengend Snippet: Clioquinol (CQ) affects the interaction between NOD-like receptor protein 3 (NLRP3) and flag-(never in mitosis gene a)-related kinase (NEK)/apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC). Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs)/human monocyte leukemia cells (THP-1) cells were incubated with different doses of CQ for 1 h and then stimulated with nigericin for 1.5 h. (A, C) Immunofluorescence assay of ASC speck. BMDMs were stained with ASC antibody (green) and 4′,6-Diamidino-2-phenylindole (DAPI) (Blue). ASC speck formation was observed by immunofluorescence. BMDMs (A), and THP-1 cells (C). (B, D) The percentage of ASC specks was measured by ImageJ (relative to LPS + nigericin). BMDMs (B), and THP-1 cells (D). (E) Western blot analysis of ASC oligomerization level after cross-linking with disuccinimidyl suberate in NP-40-insoluble pellets from BMDMs. (F) NLRP3 self-oligomerization level was measured by native-polyacrylamide gel electrophoresis (PAGE). (G, H) Immunoprecipitation analysis of the interaction between NLRP3 and NEK7 in BMDMs. IP:NLRP3 (G), and IP:NEK7 (H). (I, J) Transfected HEK-293T with plasmid overnight and treated with CQ for 8 h, and then CO-immunoprecipitation (IP) with flag antibody and Western blot was used to evaluate the interaction between green fluorescent protein (GFP)-NLRP3 and FLAG-NEK7 (I) or FLAG-ASC (J). Data are representative of n = 5 (B, D), and are expressed as mean ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, ns: not significant, determined by Mann–Whitney test (relative to LPS + nigericin). Glyceraldehyde phosphate dehydrogenase (GAPDH) served as a loading control in (E–G).

Article Snippet: Then, 500 μL of disuccinimidyl suberate (S0657, Selleck) solution (2 mM) were added to the pellets and cross-linked for 60 min at room temperature on a shaker.

Techniques: Activation Assay, Derivative Assay, Incubation, Immunofluorescence, Staining, Western Blot, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection, Plasmid Preparation, Standard Deviation, MANN-WHITNEY, Control