Fig 2: 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-1ß and TNF-a in the supernatant were measured by ELISA.(B) Immunoblot of activated Caspase-1 and mature IL-1ß in supernatants (Sup) and all NLRP3 inflammasome components in cell lysates (Lys) of BMDCs stimulated as in (A).(C) ELISA analysis of IL-1ß production in CAST knockdown THP-1 cells stimulated with silica.(D and E) ELISA analysis of IL-1ß 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-1ß 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-1ß secretion was measured by ELISA (H), and Caspase-1 activation and IL-1ß 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.

Fig 3: Burn injury induced plasma membrane recruitment of CAPN1 in the heart.A Representative confocal images. Arrows denote the positive signals of CAPN1 on the membrane. 0.5–24 h refers to the time point at which the heart specimens were harvested after burn injury. Scale bar, 10 µm. B Representative blots showing CAPN1 in the membrane fraction and the densitometric analysis results. Each independent data with the mean is presented. n = 6 rats in each group. Western blotting was performed in six independent biological experiments, and there were three technical replicates per sample. #P < 0.05, ##P < 0.01 vs. the sham group.

Fig 4: CAPN1 siRNA ameliorated burn plasma-induced cardiac cell injury.A Representative blots and densitometric analysis showing siRNA-mediated knockdown of CAPN1 and the proteolysis of a-spectrin. B Blockade of CAPN1 with siRNA improved cell viability and reduced caspase-3 activity and PARP-1 degradation. GAPDH served as a loading control. Each independent data with the mean is presented. n = 6. Western blotting was performed in six independent biological experiments, and there were three technical replicates per sample. ##, **P < 0.01.

Fig 5: CAPN2 is less efficacious in cleaving JP2 than CAPN1.(A,B) Representative Western blots (A) and summary data (B) of JP2 (mouse species) cleavage assays under different Ca2+ (CaCl2) concentrations, as indicated, using cell lysates from HEK293T cells expressing CAPN1 or CAPN2. (C,D) Western blots of calpain cleavage assays for 10 min in calpain reaction buffer. Lysates from cells overexpressing CAPNS1 in addition to CAPN1 and CAPN2 were exposed to 50 µM (C) or 500 µM (D) CaCl2, as indicated. JP2 expression and cleavage were assessed by Western blotting with anti-Flag to detect N-terminal cleavage fragments, respectively. n = 3 for each group. Data are mean ± SEM. One-way ANOVA followed by the Tukey multiple-comparisons test. * P < 0.05, *** P < 0.001.