Cells, antibodies, and other reagents
HeLa cells (human cervical cancer) were cultured in minimal essential medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. MCF7 (human breast cancer), U251 (human malignant glioblastoma), HEK 293T (human embryonic kidney), HepG2 (human hepatocellular cancer), and 4T1 (mouse breast cancer) cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. A549 (human lung cancer) cells were cultured in 10% FBS RPMI1640. Staurosporine, cycloheximide, and BAPTA-AM were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). TNFα was purchased from Sino Biological (Beijing, China). Alexa Fluor 594–conjugated wheat germ agglutinin, Alexa Fluor 594–conjugated anti-mouse IgG Ab, and Alexa Fluor 594–conjugated anti-rabbit IgG Ab were purchased from Thermo Fisher Scientific (Seoul, Korea). A23187, zVAD-fmk, and MG132 were purchased from Sigma-Aldrich (Yongin, Korea). Anti-GSDMA and anti-GSDMB Abs were purchased from ProSci (Poway, CA); anti-GSDMC, anti-GSDMD, and anti-GSDME Abs were from Proteintech (Rosemont, IL); anti-DFNB59 and anti-peflin Abs were from Novus Biologicals (Centennial, CO); anti-CD63, anti-S1PR3, anti-V5 Tag, anti-GAPDH, anti-ANXA7, anti-GFP, and anti-grancalcin Abs were from Santa Cruz Biotechnology; anti-S1PR1 Ab was from Merck Millipore (Seoul, Korea); anti-Flag M2 Ab, anti-Flag M2 affinity gel, and anti-V5 affinity gel were from Sigma Aldrich (Yongin, Korea); anti-ANXA2 Ab was from Cell Signaling Technology (Danvers, MA); anti-sorcin and anti-HD-PTP Abs were from Thermo Fisher Scientific (Seoul, Korea); and anti-CHMP4B Ab was from GeneTex (Hsinchu City, Taiwan).
Expression constructs and lentiviral transfection
CD63-pEGFP C2 was a gift from Paul Luzio (Addgene plasmid # 62964), and mCherry2-N1 was a gift from Michael Davidson (Addgene plasmid # 54517). Plasmids containing cDNAs of GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB59 were purchased from Sino-Biological (Beijing, China). The cDNAs of VPS4B, CHMP2A, CHMP3, ANXA2, ANXA7, ALG2, PEF1, sorcin, grancalcin, and HD-PTP were amplified from a mixture of HeLa cell cDNA. GSDME cDNA was amplified from a mixture of 4T1 cDNA. Constructs of cDNAs were cloned into pCDH-CMV-MCS-EF1-Puro, pCDH-CMV-MCS-EF1-Blast, pCDH-EF1-MCS-T2A-Puro, or pCDH-EF1-MCS-T2A-Blast with a C-terminal Flag tag, C-terminal V5 tag, or N-terminal Flag tag. Site-directed mutagenesis of GSDMD and GSDME was performed using a Quick-change site-directed mutagenesis kit (Thermo Fisher Scientific). For construction of GSDME fusion cDNA, a BsaBI site was introduced into the linker domain sequence of GSDME located N-terminally from the D270 caspase-cleavage site by site-directed mutagenesis, and Flag oligonucleotides, eGFP cDNA, or mCherry cDNA were cloned at the position next to S252 of GSDME cDNA using an In-Fusion HD cloning kit (TaKaRa, Seoul, Korea). All the lentiviral vectors, pGagPol (Sigma Aldrich), and pVSVg (Sigma Aldrich) were transfected into 293TN cells (System Biosciences) using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). Particles were collected on the 2nd and 3rd days after the transfection of the lentiviral plasmids and infected into the cells. Lentivirus-infected cells were puromycin- or blasticidin-selected for 2 weeks.
Cumate-inducible system for GSDME expression
Complete or partial GSDME cDNA was cloned into PiggyBac Cumate Switch Inducible vectors (PB-Cuo-MCS-IRES-GFP-EF1-CymR-T2A-Puro or PB-Cuo-MCS-3XFlag-IRES-GFP-EF1-CymR-T2A-Puro) (System Biosciences, Palo Alto, CA). The vectors were transduced into HeLa cells using the PiggyBac transposon system (System Biosciences, Palo Alto, CA), and the cells were selected for 2 weeks with 2 μg/ml of puromycin. For induction of GSDME cDNA, the cells were incubated with cumate (30 μg / ml) for 48 hr, and the expression of GSDME was confirmed by western blotting.
CRISPR/Cas9 knockout and stable cell line generation
The generation of stable knockout cell lines was achieved using the LentiCRISPRv2 system (one-vector system, Addgene #52961) or LentiGuide-Puro system (two-vector system, Addgene #52963 and #52962), which were gifts from Feng Zhang. A lentiviral plasmid containing guide RNA sequences was transfected into 293TN cells (System Biosciences) using Lipofectamine 2000 transfection reagent. Particles were collected 2 days after transfection of the lentiviral plasmids and infected into HeLa cells. The lentivirus-infected HeLa cells were puromycin-selected for 2 weeks and tested by western blotting to confirm gene knockout. Cells showing decreased or knocked-out expression of the target genes were used in further experiments.
Nanoparticle tracking analysis
Conditioned media from cell culture supernatants or plasma from EDTA-treated whole mouse blood were diluted tenfold with PBS and then analyzed for the number and size of particles using NTAs performed with a ZetaView Nanoparticle Tracking Analyzer. The following tracking parameters were used: camera sensitivity (85), shutter (250), frame rate (30f/s), minimum brightness (30), maximum area (1,000), minimum area (10), and traces (12).
LDH assay
To detect pyroptosis among apoptotic cells, we performed the lactate dehydrogenase (LDH) assay according to the instructions of the manufacturer (Biovision, Milpitas, CA), with minor modification. Briefly, the cells were cultured in 24-well plates to more than 90% confluency and then treated with staurosporine (1 μM) or TNFα (50 ng/ml) and cycloheximide (5 μg/ml) for 6 to 48 hr. The culture supernatants were collected by centrifugation of the conditioned media for 10 min at 600Xg. Cell lysates were taken from the cellular pellets after centrifugation of the conditioned media, and adherent cells were taken by lysis with 1% Triton X100 PBS. The LDH assay was performed by mixing 100 μl of the diluted supernatants or cell lysate with 100 μl of reconstituted catalyst solution in clear 96-well plates in triplicate, incubating them for 30 min at room temperature, and then measuring the absorbance at 490 nm. % cell death was calculated as follows: (absorbance of supernatant) / (absorbance of cell lysate + absorbance of supernatant) X 100.
Preparation of exosomal fractions
The conditioned media from apoptotic cells or mouse plasma diluted tenfold with PBS were centrifuged for 10 min at 200Xg and for 20 min at 2,000Xg twice to remove cellular debris and apoptotic bodies. Then the pellets were collected and washed by ultracentrifugation at 100,000Xg for 70 min twice. For fractionation of the vesicular membrane, the exosomes were incubated on ice with 100mM Na2CO3 (pH 11) for 1 hr, washed once, and resuspended in PBS.
Confocal microscopy
Cells grown on Lab-Tek four-well glass chamber slides (NUNC), were incubated in medium or medium containing staurosporine (1 μM) for the indicated times. In some experiments, the cells were incubated with Alexa Fluor 594–conjugated wheat germ agglutinin (2.5 μg/ml) for 10 min at 37 °C and washed twice with HBSS. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X100 PBS or fixed with methanol. The fixed cells were stained with the appropriate Ab and mounted with DAPI-containing mounting medium (Vector Laboratories Ltd, Peterborough, UK). Images were collected using a laser scanning confocal microscope LSM710 (Carl Zeiss, Oberkochen, Germany) equipped with argon (488 nm) and krypton (568 nm) lasers and using a x40 water immersion objective. Images were processed with ZEN 2009 light edition (Carl Zeiss).
Transmission electron microscopy (TEM) and immunogold labeling
Cells were pelleted and washed twice with PBS. Fixation was performed with phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde for 30 min at 4 °C. The pellets were rinsed twice with cold PBS, post-fixed in buffered OsO4, dehydrated in graded acetone, and embedded in Durcupan ACM resin (Fluka, Yongin, Korea). Ultrathin sections were obtained, mounted in copper grids, and counterstained with uranyl acetate and lead citrate. The specimens were observed with a Hitachi H-7600 TEM (Schaumburg, IL, USA) at 80 kV. The exosomes were resuspended in PBS, deposited onto formvar carbon-coated nickel grids for 60 min, washed with PBS, and fixed with 2% paraformaldehyde for 10 min. The exosome-coated grids were washed with PBS, transferred to a drop of the antibody, and incubated for 40 min for the immunogold labeling with anti-GSDME antibody. The grids were washed with 0.1% BSA/PBS, incubated with 10 nm-gold labeled goat-anti-rabbit IgG for 40 min, washed in PBS, and then post-fixed with 2.5% glutaraldehyde for 10 min. After washing in deionized water, the grids were stained with 2% uranyl acetate for 15 min and with 0.13% uranyl acetate and 0.4% methylcellulose for 10 min, air-dried for 5 min, and viewed by TEM.
Flow-cytometric analysis of exosomes
20 μg of exosomes were coated onto 5 μl of aldehyde/sulfate latex beads (4 μm in diameter) for 15 min at room temperature in PBS, with a final volume of 20 μl. The beads were then washed with 1 ml of PBS with shaking for 1 hr, blocked by incubation with 20 μl of FBS for 30 min, and washed thrice in PBS. The beads were resuspended in 50 μl of PBS and incubated with anti-GSDME Ab or isotype-matched irrelevant Ab for 1 hr at room temperature. After being washed thrice with PBS, the beads were incubated with PE-conjugated secondary Ab (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature. Finally, the beads were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and FlowJo software.
Preparation of cell lysates and western blots
To prepare the lysates, cells and exosome pellets were lysed in lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X100, 1 mM Na3VO4, 1 mM NaF, 1 μg/ml pepstatin A, 10 μg/ml AEBSF, 2 μg/ml aprotinin, and 1 μg/ml leupeptin), incubated on ice for 20 min, and centrifuged for 20 min to remove the supernatants. The lysates were subjected to SDS-PAGE. The proteins were then electro-transferred to PVDF membranes and incubated overnight with antibodies at 4 °C. Subsequently, the membranes were incubated with peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL, USA) for 1 h at room temperature, and the signal was detected using an enhanced chemiluminescence detection kit (Amersham Biosciences, Seongnam, Korea).
Immunoprecipitation
Cells (1 × 107 cells) were lysed using 1 ml of lysis buffer (250 mM sucrose, containing 10 mM HEPES, pH 7.4, 0.1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, 1 μg/ml pepstatin A, 10 μg/ml AEBSF, 2 μg/ml aprotinin, and 1 μg/ml leupeptin) with and without 100 μM CaCl2 for 30 min at 4 °C and then centrifuged for 20 min at 13,000 rpm at 4 °C. The supernatants were stored at −70 °C. The cell lysates were precleared with protein A/G-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) by incubation for 1 h at 4 °C with constant agitation. The precleared lysates were then incubated for overnight with anti-Flag M2 affinity agarose or anti-V5 affinity agarose at 4 °C. The immunoprecipitates were washed six times in PBS. An aliquot of each sample was subjected to western blot analysis.
4T1 orthotropic breast cancer model
All animal experiments were performed following guidelines of and protocols approved by the Laboratory Animal Research Center of Ajou University Medical Center, Suwon, South Korea. Female BALB/c mice (8 weeks old) weighing 25 g were purchased from OrientBio (Seongnam, Korea). 4T1 cells (2 X 106 cells) were injected into their mammary fat pads. Around 3 weeks after tumor injection, when the tumors had reached 1,000 mm3, the mice were injected intraperitoneally with PBS or cyclophosphamide monohydrate (BioVision, Milpitas, CA) (50 mg / kg) and doxorubicin hydrochloride (Santa Cruz Biotechnology) (3 mg / kg) twice with an interval of 24 hr. 24 hr after the last injection, the animals were sacrificed, and their livers, flushed with ice-cold PBS, and spleens were resected. The whole blood from the mice was collected by heart puncture, and the plasma was separated. The dead mice before tumors reaching 1,000 mm3 or during injection periods of anti-cancer drugs were excluded from the analysis.
Preparation of Bone marrow-derived macrophages
BMMQs were obtained from cultures of bone marrow stem cells from male BALB/c mice (6 weeks ~ 12 weeks). On day 1, the bone marrow was flushed from femurs and tibias using ice-cold PBS. The marrow plugs were disrupted into single-cell suspensions and cultured in 150-mm Petri dishes containing 25 ml of RPMI1640 supplemented with 10% FCS and M-CSF (20 ng/ml) (GenScript, Piscataway, NJ). On day 3, non-adherent cells were discarded and the culture media were replaced with fresh complete media supplemented with M-CSF. On day 6, the media were replaced. After day 7, BMMQs were kept for up to 3 weeks in culture and used for following experiments.
Real-time PCR
Total RNA was isolated using an RNeasy kit (Qiagen, Seoul, Korea). A PrimeScript RT reagent kit (TaKaRa, Seoul, Korea) was used to reverse-transcribe mRNA into cDNA. PCR was then performed on a QuantStudio 3 machine (Thermo Fisher Scientific) using SYBR Premix Ex Taq II (TaKaRa). The analysis of each sample in triplicate was performed more than twice for each experiment, and data in the figures are reported as relative quantification: average values of 2−ΔΔCT±S.D.
Measurements of intracellular free calcium ions
Intracellular free Ca2+ was measured using Calcium Sensor Dye eFluorTM 514 (eBiosciences, San Diego, CA, USA). Briefly, cells were incubated for 30 min at 37 °C in medium containing 10 μM eFluorTM 514 and then washed twice. Fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 514 nm with a FLUOstar Optima Microplate Fluorometer (BMG Labtech, Cary, NC, USA). Data are presented as relative fluorescence: MFI of treated cells / MFI of non-treated cells.
Subcellular fractionation by differential centrifugation
Preparation of the LMF and HMF was performed as described previously [46] with modifications. HeLa cells were homogenized in sucrose buffer (250 mM sucrose, 10 mM HEPES, pH 7.4, 0.1 mM CaCl2) with protease inhibitors (1 mM Na3VO4, 1 mM NaF, 1 μg/ml pepstatin A, 10 μg/ml AEBSF, 2 μg/ml aprotinin, and 1 μg/ml leupeptin) by passing them through a 21-gauge needle. The homogenates were centrifuged at 1,200 X g for 5 min to pellet the nuclear fraction and broken cellular debris. The supernatants were further centrifuged at 15,000 X g for 10 min to separate the HMF and post-nuclear supernatants. The post-nuclear supernatants were then centrifuged for 1.5 h at 130,000 X g to isolate the LMF and cytosolic fraction.
Statistical analyses
The values are presented as the mean ± S.D. Statistical analyses were performed using one-way analysis of variance (ANOVA) with either all pairwise multiple comparisons or multiple comparisons versus control (Holm-Sidak method). Statistical significance was considered at the P < 0.05 level.