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: ZBTB18 is cleaved by calpain(A) List of protease coding genes, which are differentially expressed between BTSCs with or without ZBTB18 SF. CAPN2 appears to be significantly more expressed in BTSCs with ZBTB18 SF.(B and C) Box-plot representation of CAPN2 expression extrapolated from RNAseq analysis (B) and from quantitative RT-PCR (C) of BTSC lines with or without ZBTB18 SF. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; crosses represent sample means; width of the boxes is proportional to the square root of the sample size. n = 3, five sample points (B) and n = 4, five sample points (C).(D and E) Western blot analysis (D) and relative quantification (E) of CAPN2 and ZBTB18 levels in LN229 GBM cells and different BTSCs characterized by the presence (marked in orange) or absence (mrked in blue) of ZBTB18 SF. Both ZBTB18 FL and SF were detected with a ZBTB18 total antibody (Abcam).(F) Western blot analysis of ZBTB18 expression, using a ZBTB18 total antibody (Abcam) in U3082 cells treated with or without different concentrations of calpain inhibitor (PD150606) for 16 h.(G) Western blot analysis of ZBTB18 expression, detected with a ZBTB18 total antibody (Abcam), in U3082 cells treated with the calpain inhibitor (PD150606, 100µM) in a time-course experiment.(H) Western blot analysis of CAPN2 and ZBTB18 expression (FL and SF) in U3082 cells on CAPN2 knockdown with two independent shRNAs. ZBTB18 was detected with a ZBTB18 total antibody (Abcam).(I and J) Anti-FLAG (mouse, Sigma) Western blot analysis (I) and relative quantification (J) of ZBTB18 FL and SF expression in SNB19 cells transduced with ZBTB18 FL, on treatment with cycloheximide (100 µg/mL) for several time points. The level of individual ZBTB18 high and low bands was quantified using the ImageJ software (Bio-Rad) and normalized to aTubulin.(K and L) Western blot analysis using anti-FLAG antibody (mouse, Sigma) (K) and relative quantification (L) of ZBTB18 FL and SF expression in SNB19 cells transduced with empty vector (EV) or with ZBTB18 FL, and exposed to MG132 (5 µM) or DMSO, for the indicated time points. The quantification of Western blot bands was performed using the ImageJ software (Biorad) and normalized to aTubulin. Either individual ZBTB18 FL and SF (left panel) or total ZBTB18 levels (right panel) were plotted. The asterisks indicate unspecific bands. See also Figure S2.

Fig 4: Transverse aortic constriction (TAC) induces cardiac Calpain expression and JP2 cleavage.(A) Representative Western blots of JP2, CAPN1, CAPN2 and CAPNS1 in heart homogenates from sham and TAC operated animals. (B–D) Western blot quantification of full-length JP2 relative to GAPDH loading control (B) and its ~75 kD N-terminal (C) and 25 kD C-terminal (D) cleavage products normalized to full-length JP2. (E–G) Quantification of CAPN1 (E), CAPN2 (F) and CAPNS1 (G) expression in TAC and Sham hearts normalized to GAPDH. n = 6 for each group. Data are mean ± SEM. One-way ANOVA followed by the Tukey multiple-comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., non-significant.

Fig 5: Human JP2 (hJP2) is cleaved by CAPN1 and CAPN2 in the same region analogous to mouse JP2 R565/T566.(A) Schematic of Flag- and HA-tagged human JP2 deletion constructs used to test calpain cleavage site selection. (B–E) Western blots of in vitro calpain assays. HEK293T cells were co-transfected with hJP2-WT, hJP2?486–493 or hJP2?570–575 and either CAPN1 (B,D) or CAPN2 (C,E) cDNAs for 24 h. Cell lysates were exposed with and without 50 µM or 2 mM Ca2+ for 20 min in calpain reaction buffer in the absence or presence of 10 µM MDL-28170. Human JP2 expression and cleavage were assessed with anti-Flag or anti-HA antibodies to detect N- and C-terminal cleavage fragments, respectively.