Fig 1: MPP+-induced inflammation of HMC3 microglia and anti-inflammatory potentials of NC009 compounds. (A) Cytotoxicity of MPP+ against HMC3 cells by MTT and NO release assays. Cells were treated with MPP+ (0–10 mM) and cell viability and NO release in culture medium was measured the following day (n = 3). To normalize, the relative cell viability in MPP+-untreated (inactive) cells was set at 100%. p values: comparisons between inactive and activated cells (* p < 0.05, *** p < 0.001). (B) Experimental flow chart. HMC3 cells were plated on day 1. After 20 h, cells were pre-treated with test compounds (1–10 µM) for 8 h, followed by MPP+ (3 mM) treatment for 20 h. On day 3, the HMC3 cells were examined for cell viability (MTT assay) and NO release (Griess assay) in culture medium. In addition, cells with 10 µM compound treatment were examined for CD68 and CD11B expression (ICC staining), IL-1ß, IL-6, TNF-a release (ELISA), and NLRP3, CASP1, iNOS, IL-1ß, IL-6, and TNF-a expression (Western). (C) Cell viability and NO release assays (n = 3). The relative cell viability in MPP+-untreated cells was normalized as 100%. (D) CD68 and CD11B expression (n = 3). Cells were analyzed by immunofluorescence using antibodies against CD68 (yellow) and CD11B (green). Cell nuclei were counterstained with DAPI (blue). (E) IL-1ß, IL-6, and TNF-a release assays (n = 3). (F) NLRP3, CASP1, iNOS, CREB, IL-1ß, IL-6, and TNF-a levels analyzed by immunoblotting using GAPDH as a loading control (n = 3). To normalize, protein expression level in MPP+-untreated cells was set at 100%. (C–F), p values: comparisons between MPP+-untreated and treated cells (# p < 0.05, ## p < 0.01, ### p < 0.001), or with and without compound addition (* p < 0.05, ** p < 0.01, *** p < 0.001).