TMAO activated NLRP3 via the mitochondrial ROS. (A) Representative image of BMDMs labeled with MitoSOX for mitochondrial ROS detection (green, Mito; red, MitoSOX; blue, Hoechst). Scale bar, 10 μm. (B-E) Mean fluorescence intensity (MFI) of MitoSOX determined by ImageJ (B; N = 5 in each group; **P < .01) and percentage of fluorescence-expressing MitoSOX determined by FACS analysis (C) in TMAO-treated BMDMs. Mitochondrial membrane potential as the ratio of JC-1 red to JC-1 green was detected by immunofluorescence (D; scale bar, 10 μm), and dysfunctional mitochondrial respiration labeled with MitoTracker Green^highMitoTracker Red^low was detected by FACS analysis (E) in BMDMs treated with 300 μM TMAO for 24 hours. (F) Representative immunofluorescence staining of NLRP3 (green, Alexa Fluor 488), ASC (red, Alexa Fluor 594), DAPI (blue) in TMAO-treated or TMAO+Tempo–treated BMDMs. Scale bar, 5 μm. (G) Western blotting analysis of NLRP3, IL-1β, and cleaved IL-1β in TMAO-treated or TMAO+Tempo–treated BMDMs. (H) Secreted IL-1β in BMDM-cultivated supernatants with TMAO, Tempo, or TMAO+Tempo. N = 4 in each group. **P < .01. (I-J) Caspase-1 activity determined as pNA concentration (I; N = 4 in each group; *P < .05, **P < .01) or fluorescein isothiocyanate–FLICA percentage (J) was analyzed in TMAO-treated or TMAO+Tempo–treated BMDMs. (K) Expression of TNF-α (N = 3 in each group) and IL-6 (N = 4 in each group) was determined by ELISA in BMDM supernatants cultured with TMAO, Tempo, TMAO+Tempo for 24 hours. **P < .01. (L) Expression of CXCL9 and CXCL10 in BMDMs was determined by RT-PCR after being treated with Tempo, TMAO, or TMAO+Tempo for 24 hours. N = 3 in each group. **P < .01.