Fig. 2: Necrotic TECs activate NLRP3 inflammasome in macrophages.

PMA-differentiated THP-1 cells were treated with or without 100 ng/ml LPS for 3 h before incubated with different concentrations of conditioned medium (CM, primary concentration; CM 1:1, 1:1 dilution with culture medium; CM 1:2, 1:2 dilution with culture medium) for 6 h, incubated with 5 mM ATP for 1 h (positive control) or incubated with medium from normal NRK-52E cells (Med) as control. Culture supernatants (Sup) and cell lysates (Lys) were collected. Representative immunoblot analyses of the immature (Pro-) and mature forms of caspase-1 and IL-1β in culture supernatants and cell lysates (A). Quantification of IL-1β in supernatant under different treatments by ELISA (B). Relative mRNA expressions of NLRP3, AIM2, NLRP1 and NLRC4 in THP-1 cells (C). Representative immunofluorescence images of ASC in THP-1 cells. Arrows: ASC specks (scale bar, 20 μm) (D). Representative immunoblot analysis of the immature (Pro-) and mature forms of caspase-1 and IL-1β in culture supernatants and cell lysates from THP-1 cells, Def-NLRP3-THP-1 cells, and Def-ASC-THP-1 cells (E). Quantification of IL-1β in supernatant of THP-1 cells under different treatments by ELISA (F). Data are presented as the mean ± SD of the representative experiment performed in at least three biological replicates. The p values were calculated using one-way ANOVA test. *p < 0.05; **p < 0.01; ***p < 0.001.