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. 2020 Nov 18;11:581165. doi: 10.3389/fimmu.2020.581165

Figure 4.

Figure 4

Flagellated bacteria-released outer membrane vesicles trigger NLRC4-dependent inflammasome activation. (A) Immunoblots from mouse BMDMs treated with OMV (0.5 or 3 µg/ml) isolated from Pseudomonas aeruginosa PAO1 for 8 h. (B) Immunoblots from Nlrc4 +/+ or Nlrc4 −/− mice BMDMs treated with P. aeruginosa OMV (1 or 7 µg/ml) for 8 h. (C) Immunoblots from Nlrp3 +/+ or Nlrp3 −/− mice BMDMs treated with P. aeruginosa OMV (5 µg/ml) for 8 h. (D, E) Quantification of IL-1β in the culture supernatants of Nlrc4 +/+ or Nlrc4 −/− (D) or Nlrp3 +/+ or Nlrp3 −/− (E) mice BMDMs treated with P. aeruginosa OMV (1 or 7 µg/ml, D; 5 µg/ml, E) for 8 h, or treated with Pam3CSK4 (1 µg/ml, 3 h), followed by the transfection of LPS (2 µg/ml, D; 1 µg/ml, E) for 6 h. (n = 3) (F) Immunoblots from Nlrp3 +/+ or Nlrp3 −/− mice BMDMs treated with E. coli BL21 or DH5α OMVs (5 µg/ml) for 8 h. (G) Immunoblots from Nlrc4 +/+ or Nlrc4 −/− mice BMDMs treated with E. coli BL21 OMVs (1 or 5 µg/ml) for 8 h. (H, I) Quantification of IL-1β in the culture supernatants of Nlrp3 +/+ or Nlrp3 −/− (H) or Nlrc4 +/+ or Nlrc4 −/− (I) mice BMDMs treated with E. coli BL21-derived OMVs (5 µg/ml, H; 1 or 5 µg/ml, I) or DH5α-derived OMVs (5 µg/ml, H) for 8 h, or treated with Pam3CSK4 (1 µg/ml, 3 h), followed by the transfection of LPS (1 µg/ml, H; 2 µg/ml, I) for 6 h. (n = 3) Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with the group in the Nlrp3 +/+ cells. (*P < 0.05, ***P < 0.001, n.s. not significant).