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. 2017 Sep 4;6(9):e006347. doi: 10.1161/JAHA.117.006347

Figure 2.

Figure 2

Trimethylamine‐N‐oxide (TMAO)‐induced inflammation via nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3) inflammasome activation in endothelial cells. A, Cells were treated with TMAO at a series of concentrations (150, 300, 600, and 900 μmol/L) for 24 hours, and the expression of NLRP3 and caspase‐1 p20 was detected via Western blot. B, Bar charts showing quantification of endogenous NLRP3 and caspase‐1 p20. C, Cells were incubated with 600 μmol/L TMAO for different time intervals (4, 8, 12, and 24 hours), and the expression of NLRP3 and caspase‐1 p20 was detected via Western blot. D, Bar charts showing quantification of endogenous NLRP3 and caspase‐1 p20. Cells were treated as described for A and B. Thereafter, caspase‐1 activity was measured using caspase‐1 activity kits (E and F). G, Cells were pretreated with YVAD (10 μmol/L) or MCC950 (10 μmol/L) for 2 hours, and then exposed to TMAO (600 μmol/L) for a further 24 hours. Expression of IL‐1β, ICAM‐1, MMP‐9, and caspase‐1 p20 was detected via Western blot. H, Bar charts showing quantification of the indicated proteins. I, Human umbilical vein endothelial cells (HUVECs) were transfected with NLRP3 siRNA as described in Materials and Methods. After 24 hours, cells were incubated with TMAO (600 μmol/L) for 24 hours, and the expression of IL‐1β, ICAM‐1, MMP‐9 and Casp1 p20 was detected via Western blot. J, Bar charts showing quantification of indicated proteins. Values are expressed as means±SE (n=3). a P<0.05, b P<0.01 vs vehicle‐treated control group; c P<0.01 vs TMAO‐treated group; AU indicates arbitrary units; ICAM, intercellular adhesion molecule; IL, interleukin; MMP, matrix metallopeptidase.