Fig. 4 A-C.

Priming of the NLRP3 inflammasome depends on adherent PAMPs at the mRNA (A-B) and protein (C) levels. Wild-type macrophages were treated with either endotoxin-free titanium particles (A-B, green squares) or titanium particles with adherent bacterial debris (A-B, red circles). Markers of priming (NLRP3 and IL1β mRNA and pro-IL1β protein) were measured in cell lysates at indicated time points and the mRNA values were normalized to GAPDH mRNA. N = 3 independent experiments were performed (A-B). Each experiment included triplicate cell culture wells per group, each assayed in triplicate. Error bars represent SD. Statistical significance was determined by one-sided analysis of variance. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 with comparisons made between titanium particles with adherent bacterial debris and endotoxin-free titanium particles at each indicated time point. Pro-IL1β protein was detected by Western blot (C). The pictured gel is representative of three independent experiments. Ti = titanium; NLRP3 = Nod-like-receptor-protein-3; mRNA = messenger RNA; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.