Figure 1. Calpain Activation Plays an Important Role in NLRP3 Inflammasome Activation.

(A) BMDCs were pretreated with calpain inhibitors calpeptin (30 μM) or PD150606 (50 μM) for 30 min and then activated with different crystals for 5 hr. IL-1β and TNF-α in the supernatant were measured by ELISA.

(B) Immunoblot of activated Caspase-1 and mature IL-1β in supernatants (Sup) and all NLRP3 inflammasome components in cell lysates (Lys) of BMDCs stimulated as in (A).

(C) ELISA analysis of IL-1β production in CAST knockdown THP-1 cells stimulated with silica.

(D and E) ELISA analysis of IL-1β production in CAPN1- or CAPN2-knockdown (D) or CAPN1-knockout THP-1 cells (E, generated with 2 different small guide RNAs).

(F) Immunoblot of activated Caspase-1 and mature IL-1β in supernatants and all NLRP3 inflammasome components in cell lysates of CAPN1-deficient or wildtype (WT) THP-1 cells.

(G) CAPN1-deficient or WT THP-1 cells were treated with 3 μm biotin-coated beads for 30 min and then the cells were stained with Alexa 405-strepavidin without permeabilization to distinguish adherent or complete engulfed beads. Percentages of complete phagocytosis of 100 cells were plotted.

(H and I) BMDCs were pretreated with calpeptin or PD150606, then treated with ATP or nigericin. IL-1β secretion was measured by ELISA (H), and Caspase-1 activation and IL-1β processing were detected by immunoblotting (I).

In (A), (C)–(E), and (H), data are shown as mean ± SEM of n = 3. All results are representative of 2 or 3 independent experiments. See also Figure S1.