Fernandes-Alnemri et al.
Generation of Stable THP-1-ASC-GFP Cell Line. The monocytic cell line THP1 was cultured in RPMI 1640 supplemented with 10 mM N- (2-hydroxyethyl) piperazine-N’- (2-ethanesulfonic acid), 1 mM sodium pyruvate, 55 µM b-mercaptoethanol, 10 % fetal bovine serum and 200 µg·ml–1 penicillin and 100 µg·ml–1 streptomycin sulfate. Stable THP-1 cells expressing ASC-GFP fusion protein were generated by retroviral gene transfer with recombinant MSCV expression vectors. The amphotropic packaging cell line Phoenix (G.P. Nolan’s laboratory, Stanford University Medical Center, Stanford, CA) was transfected pMSCVpuro-ASC-EGFP-N1 plasmid using the LipofectAMINE transfection method. Forty-eight hours after transfection, the GFP expressing cells were sorted three times over a period of 4 weeks by flow cytometry until more than 95% of the cells is stable GFP positive. To infect THP-1 cells, the stable Phoenix cells were seeded in THP-1 culture medium for 24 h and then the culture supernatants containing retroviral particles were collected and filtered through 0.45 m membrane. THP1 cells (1 x 106 cells/well) were then centrifuged in 6-well plate for 60 min at 2500 rpm at 32°C in the presence of 3 ml of retrovirus-enriched culture supernatant supplemented with 4 µg/ml of polybrene (Sigma). Plates were placed back in a CO2 incubator at 37°C for 2 h. Fresh THP-1 medium was then added and the cells were allowed to recover for 24 h. The cells were subjected to another cycle of infection and then allowed to recover for 72h before sorting by flow cytometry. The GFP expressing cells were sorted three times over a period of 4 weeks until more than 95% of the cells is stable GFP positive. The stable expression of ASC-GFP was verified by western blotting and functional assays.
Generation of Stable HEK293T Cell Lines. HEK293T cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum, 200 µg·ml–1 penicillin and 100 µg·ml–1 streptomycin sulfate. 293-caspase-1, and 293- cells have been described before 1. To generate the 293-caspase-1- ASC-EGFP-N1 (293-C1-ASC-GFP) and 293-caspase-1-EGFP-N1 (293-C1-GFP) stable cell lines, the parental 293-caspase-1 cells were transfected with pEGFP-N1-ASC 1 or pEGFP-N1 plasmids respectively. The stable GFP expressing cells were then sorted three times over a period of 4 weeks by flow cytometry until more than 95% of the cells were GFP positive.
Purification of ASC pyroptosomes from LPS-stimulated macrophages and chemical cross-linking. THP-1 cells were treated with PMA and seeded in 10 cm dishes. Three hours after PMA treatment the medium containing PMA was replaced with fresh medium and the cells were allowed to attach to the plates overnight. Next day cells were preincubated with zVAD-FMK (50 µM) for 30 min and then treated with LPS (5 µg/ml) for 3h. Cells were harvested and then lysed in buffer A (20 mM Hepes-KOH, pH7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose). The cell lysate was centrifuged in 1.5 ml Eppendorff tubes at 1500 rpm to remove the bulk nuclei and the resulting supernatant was diluted 2X with buffer A and then filtered using a 5 micron filter to remove any remaining nuclei. The supernatant was diluted with one volume CHAPS buffer (20 mM Hepes-KOH, pH 7.5, 5 mM MgCl2, 0.5 mM EGTA, 0.1 mM PMSF, 0.1 % CHAPS) and then centrifuged at 5000 rpm to pellet the ASC pyroptosomes. The crude pellet was resuspended in CHAPS buffer, and either was chemically cross-linked using the non-cleavable Disuccinimidyl suberate (DSS) cross linker (4 mM) for 30 minutes, or subjected to further purification. To further purify the ASC pyroptosomes, the crude 5000 rpm pellet from above was resuspended in CHAPS buffer, layered over a 40% percoll cushion and centrifuged at 14000 rpm in an Eppendorff table top centrifuge. The pelleted ASC pyroptosomes at the bottom of the 40% percoll cushion were then washed in CHAPs buffer and centrifuged again at 12,000 rpm. The purified ASC pyroptosomes were resuspended in CHAPS buffer and used for different assays. ASC pyroptosomes were isolated from bone marrow derived mouse macrophages after stimulation with LPS (3 h) followed by ATP (1 h) in the absence of zVAD-FMK and then cross-linked as described above.
In vitro assembly and purification of ASC pyroptosomes from THP-1 cell lysates. THP-1 cell pellets were lysed in CHAPS buffer and then centrifuged at 14,000 rpm to obtain crude lysates. S100 lysates were prepared from the crude lysates by centrifugation at 100,000 xg. ASC pyroptosomes were in vitro assembled by incubation of S100 lysates at 37° C for 30 min. The assembled ASC pyroptosomes were pelleted from the lysates by centrifugation at 5000 rpm. The pellets were resuspended in CHAPS buffer, layered over a 40% percoll cushion and centrifuged at 14,000 rpm. The pelleted ASC pyroptosomes at the bottom of the 40% percoll cushion were then washed in CHAPs buffer and centrifuged again at 12,000 rpm. The purified ASC pyroptosomes were resuspended in CHAPS buffer and used for different assays.
In vitro caspase-1 activation and IL-1 cleavage Assays. Lysates containing Flag-tagged procaspase-1 (WT and C/A) and untagged pro-IL-1 were isolated from 293-caspase-1 or 293-pro-IL-1 stable cell lines in CHAPS buffer. Purified ASC pyroptosomes were incubated with procaspase-1 lysates together with pro-IL-1 in CHAPS buffer for different periods of time at 37° C. The reaction mixtures were then fractionated by SDS-PAGE and analyzed by western blotting with anti-Flag and anti-IL-1 antibodies. In some experiments, procaspase-1 was purified from 293-caspase-1 lysates using anti-Flag-agarose beads (Sigma) and eluted with Flag peptide according to the manufacturer’s protocol.
Expression and purification of recombinant ASC from bacteria. WT ASC or chimeric ASC proteins were expressed in bacteria with N-terminal His-tag using the pET-28a vector. ASC was purified by standard procedures using Talon affinity beads.
Generation of Antibodies. The anti-human caspase-1 polyclonal antibody was raised in rabbits against a bacterially produced protease domain (p30) of human caspase-1 (amino acids 120-402). The anti-IL-1 monoclonal antibody (32D) was obtained from the NCI preclinical repository, Biological resource branch. Anti human and mouse ASC antibodies were obtained from J. Sagara (Japan). Anti-mouse caspase-1 antibody was a gift from Junying Yuan. Anti-human cryopyrin monoclonal antibody (anti-NALPy-3b) was obtained from Alexis.
1. Yu, JW, Wu, J, Zhang, Z, Datta, P, Ibrahimi, I, Taniguchi, S et al., (2006) Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization. Cell Death Differ 13: 236-49.
Supplementary Figure 1. Caspase-1 and ASC are required for induction of pyroptosis in 293 cells. The indicated 293 stable cell lines were left untreated (left panels) or treated with PMA to induce formation of ASC pyroptosomes. Cells were then observed by confocal microscopy (63x magnification). Notice the pyroptotic phenotype induced by ASC pyroptosome in the 293-C1-ASC-GFP cells, which express both procaspase-1 and ASC-GFP, but not in the 293-ASC-GFP, which express only ASC-GFP, or 293-C1-GFP, which express only procaspase-1 and GFP.
Supplementary Movie 1. Time-lapse movie of LPS-treated THP-1-ASC-GFP cells. Nuclear stain is seen in the blue window. ASC-GFP is seen in the green window. Mitochondrial mitotracker red is seen in the red window. Phase contrast is seen in the gray window. Images were recorded every 17.5 seconds at 63X magnification. Movie contains 147 images.
Supplementary Movie 2. Merged RGB-gray images of the movie shown in Supplementary Movie 1.
Supplementary movie 3. Time-lapse movie of a larger field of LPS-treated THP-1-ASC-GFP cells. This one color movie shows merged ASC-GFP and gray phase contrast images. Notice how cells explode after formation of the ASC pyroptosome (green cluster) to release their contents in the extracellular space, and then the plasma membrane reseals and swells to form a balloon around the condensed nucleus.
Supplementary Movie 4. Time-lapse movie of zVAD-FMK and LPS-treated THP-1-ASC-GFP cells. Nuclear stain is seen in the blue window. ASC-GFP is seen in the green window. Mitochondrial mitotracker red is seen in the red window. Phase contrast is seen in the gray window. Images were recorded every 17.5 seconds at 63X magnification. Movie contains 147 images.
Supplementary Movie 5. Merged RGB-gray images of the movie shown in Supplementary Movie 4.