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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: J Immunol. 2013 Jul 31;191(5):2665–2679. doi: 10.4049/jimmunol.1202733

Figure 6.

Figure 6

NLRP3 inflammasome in KCs is activated by extracellular histones through TLR9 pathway. Whole cell lysate from KCs after stimulation was subjected to western blot analysis of activated caspase-1, IL-1β and IL-18. For Western blot images: The blots shown are representatives of three experiments with similar results. (A) Cultured mouse KCs were exposed to hypoxia (1% O2) from 0 to 12 hours. (B) The activated caspase-1 in cultured KCs obtained WT mice stimulated with normoxia or hypoxia (1% O2) overnight was visualized with caspase-1 fluorochrome inhibitor of caspase-1 reagent and observed under confocal microscope. Green, actin; blue, nuclei; red, activated caspase-1. (C) Cultured KCs from WT or TLR9 mutant mice were stimulated with exogenous histones from dosage of 0 to 50 μg/mL for 12 hours. (D) The activated caspase-1 in cultured KCs that were stimulated with exogenous histones (25 μg/mL) or TLR9 agonist (15μg/mL) or PBS for 12 hours, was visualized with caspase-1 fluorochrome inhibitor of caspase-1 reagent and observed under confocal microscope. Green, actin; blue, nuclei; red, activated caspase-1. (E) Cultured mouse KCs were stimulated with TLR9 agonist from dosage of 0 to 15μg/mL for 12 hours. (F) Quantitation of activated caspase-1 in cultured mouse KCs was measured using the analytical software MetaMorph™ and normalized to nuclei within the sample field. ANOVA Holm-Sidak method, *P < 0.05; Histones or TLR9 agonist vs. PBS treated-KCs, **P < 0.05; Hypoxia vs. Normoxia treated KCs. (G) Cultured mouse KCs were stimulated with TLR9 antagonist or/and exogenous histones for 12 hours.