ASC activation induces apoptosis or necrosis in various tumor cell lines. A, ASC and β-actin levels were examined by Western blotting. B–E, the indicated cell lines were transduced with a lentiviral vector expressing C12N2. The cells were then treated with or without MDP (100 ng/ml). B, cell morphology was examined under a microscope. Representative necrotic and apoptotic cells are shown by arrows and arrowheads, respectively. Scale bars, 20 μm. C, leukemia and melanoma cell lines were stained with PI and Cy5-annexin V, and the percentages of apoptotic (Apo) and necrotic (Nec) cells were determined by flow cytometry. The staining profiles are shown in supplemental Fig. S1. D, pancreatic cancer cell lines were stained with Hoechst 33342 and PI and examined under a fluorescence microscope. Necrotic cells (arrows) had morphologically normal nuclei that were stained with PI. Scale bar in the top left panel, 20 μm. E, apoptotic and necrotic cells were counted under a fluorescence microscope. At least 500 total cells were counted for each group. Error bars in C and E indicate S.D.

The RIP1-RIP3 axis is not involved in ASC-mediated necrosis. A, the RIP3 (RIPK3) and β-actin (ACTB) mRNA levels in the indicated tumor cell lines were analyzed by RT-PCR. B, THP-1 cells (left) or C12N2-expressing THP-1 cells (right) were pretreated with necrostatin-1 (10 μm) for 1 h. Then, the THP-1 cells were treated with recombinant mouse TNF-α (30 ng/ml) and Z-VAD-FMK (40 μm) for 48 h, whereas the C12N2-expressing THP-1 cells were treated with MDP (100 ng/ml) for 10 h. Cell viability was assessed by WST-1 assay as described in the legend for Fig. 2. Error bars indicate S.D.

Caspase-1 is required for ASC-mediated necrosis. A, CLC12N2-Nec cells were transfected with control (siCont, circles) or caspase-1-targeting siRNA (siCasp1, triangles) 1–3 times at 3-day intervals. At 3, 6, or 9 days after the first transfection, the cells were treated with MDP (100 ng/ml) for 12 h, and the proportions of apoptotic (Apo, open symbols) and necrotic cells (Nec, closed symbols) were determined by flow cytometry as described in the legend for Fig. 1. Caspase-1 (Casp1) and GAPDH expression levels at the indicated times before MDP stimulation were examined by Western blotting (lower panels). B, NUC12N2 and NU-GFP-Casp1 cells (NU, Casp1) were treated with MDP (1000 ng/ml) or left untreated for 6 h and then stained with Hoechst 33342 and PI and examined under a fluorescence microscope. The upper and lower panels show Hoechst- and PI-stained images, respectively. Apoptotic cells had pyknotic nuclei that were brightly stained with Hoechst 33342 but not with PI, whereas necrotic cells had morphologically normal nuclei that were stained with PI. Scale bar, 20 μm. GFP-caspase-1 (GFP-Casp1) and GAPDH expression levels in these cells before MDP stimulation were examined by Western blotting (lower panels). C, NOMO-1 cells treated with control or ASC-targeting siRNA (siASC) for 72 h were transduced with empty lentiviral vector (Vector) or with the vector expressing wild-type (wt) NLRP3 or its Y570C mutant (mt). Twelve hours after transduction, cytotoxicity was assessed by lactate dehydrogenase (LDH) release (upper panel). ASC and GAPDH expression levels were examined by Western blotting 48 h after the siRNA transfection (lower panels). D and E, NOMO1-shCont (shCont) and NOMO1-shCasp1 (shCasp1) cells were transduced with an empty lentiviral vector or one expressing the wild type or Y570C mutant of NLRP3 and were cultured for 12 h. D, cytotoxicity was assessed as described in C. Caspase-1 and GAPDH levels were examined by Western blotting (lower panels). E, cells were examined as described in B. Scale bar, 20 μm. Arrowheads indicate apoptotic cells. F, NOMO1-shCont and NOMO1-shCasp1 cells were pretreated with Z-IETD-FMK (40 μm) or dimethyl sulfoxide (DMSO) (solvent control) for 1 h. Cells were then transduced with a lentiviral vector expressing the Y570C mutant of NLRP3 and were further cultured for 12 h. Apoptotic and necrotic cells were counted under a fluorescence microscope. Error bars in A, C, D, and F indicate S.D.

Caspase-1, but not its catalytic activity, is required for the necrotic cell death of monocytes infected with bacteria. A and G, optical phase contrast images of NOMO-1 cells either uninfected (left panels) or infected with S. aureus (A, right panel) or P. aeruginosa (G, right panel) at an m.o.i. of 20 for 4 h. Scale bar, 20 μm. B and H, NOMO-1 cells were transfected with control (Cont), ASC-, NLRP3-, or NLRC4-targeting siRNA. After 72 h, the cells were infected with S. aureus (B, m.o.i. 20 or 50) for 2 h or with P. aeruginosa (H, m.o.i. 50) for 4 h. The knockdown efficiency was examined by RT-PCR (B, right panels). LDH, lactate dehydrogenase. C, NOMO-1 cells were pretreated with 20 μm of the indicated inhibitors for 1 h. Cells were then infected with S. aureus for 2 h. D and J, NOMO-1 cells were pretreated with the indicated concentrations of Ac-YVAD-CMK for 1 h. The cells were then infected with S. aureus (m.o.i. 20) for 2 h (D) or with P. aeruginosa (m.o.i. 50) for 4 h (J). E and I, NOMO1-shCont (shCont) and NOMO1-shCasp1 (shCasp1) cells were infected with S. aureus (m.o.i. 50) for 2 h (E) or P. aeruginosa (m.o.i. 50) for 4 h (I). F, NOMO-1 cells were transfected with control (Cont) or caspase-1-targeting siRNA (C1) 1–3 times at 4-day intervals. At 4, 8, or 12 days after the first transfection, the cells were infected with S. aureus (m.o.i. 20) for 2 h. Caspase-1 and GAPDH levels at the end of a 4-, 8- or 12-day culture before infection were examined by Western blotting. B–F and H–J, cytotoxicity was assessed by lactate dehydrogenase release assays. The amount of IL-1β in culture supernatants was examined by ELISA. Percentage of control = (value of cells treated with Ac-YVAD-CMK or caspase-1-targeting siRNA/value of cells treated with the solvent control or control siRNA) × 100. Error bars in B–F and H–J indicate S.D.

Effect of protease inhibitors on ASC-mediated cell death. A, NUGC-4, HMV-II, and G-361 cells expressing C12N2 were left untreated or were treated with 40 μm of Z-IETD-FMK or CA-074Me for 1 h and were then cultured with or without MDP (100 ng/ml) for 12 h. The proportion of apoptotic cells was determined as described in the legend for Fig. 1. B, COLO205, THP-1, NOMO-1, and SK-MEL-28 cells expressing C12N2 were pretreated with the indicated inhibitors (20, 40 and 80 μm) for 1 h and then cultured with or without MDP (100 ng/ml) for 8 h. Cell viability was assessed by WST-1 assay. Cell death (percentage) = {(A₄₅₀ of cell culture without MDP − A₄₅₀ of cell culture with MDP)/A₄₅₀ of cells cultured without MDP} × 100. Error bars in A and B indicate S.D.

Caspase-1 expression correlates with the necrotic phenotype. A, CLC12N2-Apo and CLC12N2-Nec cells were treated with MDP (100 ng/ml) for 12 h and then analyzed as described in the legend for Fig. 1. Nec-type, necrosis-type cells; Apo-type, apoptosis-type cells. B, CLC12N2-Apo and CLC12N2-Nec cells were pretreated with Z-IETD-FMK (5, 10, 20, or 40 μm) for 1 h or left untreated and then were treated with MDP (100 ng/ml) for 12 h. The percentages of apoptotic and necrotic cells were determined by flow cytometry. C, gene expression profiles of CLC12N2-Nec and CLC12N2-Apo cells or COLO205 and NUGC-4 cells were compared by DNA microarray analyses. The top 10 genes with greater expression in CLC212N2-Nec cells than in CLC12N2-Apo cells are listed in the upper panel, whereas those expressed more in CLC12N2-Apo cells than in CLC12N2-Nec cells are shown in the lower panel. Numbers shown are the -fold difference in gene expression levels between the indicated cell lines. Original datasets of the microarray analyses are available at the Center for Information Biology Gene Expression (CIBEX) database (accession number CBX131). D, the caspase-1 mRNA levels relative to that of β-actin, as determined by quantitative RT-PCR, are shown for the indicated cell lines. E, caspase-1 and β-actin protein levels in the indicated cells were examined by Western blotting. Error bars in B and D indicate S.D.

Caspase-1 catalytic activity is not required for ASC-mediated necrosis. A, SK-MEL-28 cells stably expressing C12N2 (SKC12N2) were treated with MDP (1000 ng/ml) for the indicated period. The large subunit of mature caspase-1 (p20) in cell lysates and culture supernatants (Sup) was detected by Western blotting. B, SKC12N2 cells were left untreated or were pretreated with Ac-YVAD-CMK (40 μm) and then stimulated with MDP (1000 ng/ml) for 4 h. Cytotoxicity was assessed by a lactate dehydrogenase (LDH) release assay, and caspase-1 p20 in culture supernatants was detected by Western blotting. C and D, NUC12N2 cells were transduced with a lentiviral vector expressing GFP, wild-type caspase-1, or the C285S mutant of caspase-1. C, caspase-1 and β-actin levels in the indicated cells were examined by Western blotting. D, NUC12N2 cells expressing GFP, wild-type caspase-1 (WT), or the C285S mutant of caspase-1 (CS) were cultured with MDP (1000 ng/ml) for 6 h and then stained with Hoechst 33342 and PI. Apoptotic and necrotic cells were counted under a fluorescence microscope. E, the cells described in D were further transfected with a pro-IL-1β-expressing plasmid. After 21 h, the cells were treated with MDP (1000 ng/ml) for 3 h, and IL-1β in the culture supernatant was measured by ELISA. Error bars in B, D, and E indicate S.D.