Primary PDLF and DPC culture
Periodontal ligament and dental pulp tissues were obtained from freshly extracted intact premolars or third molars. The teeth were washed with phosphate-buffered saline (PBS, Gibco, Grand Island, NY, USA) containing 2% penicillin‒streptomycin solution (HyClone, Logan, UT, USA). Periodontal ligament tissues were collected from the middle third of roots using a scalpel. Then, the teeth were split with a hammer. Pulp tissues were collected from the pulp chamber. Collected tissues were cut into pieces with scissors and cultured in α-minimum essential medium (αMEM; Gibco) containing 20% fetal bovine serum (FBS; Gibco) in a 37 ℃ incubator with 5% CO2 until subculture. Beginning with passage 2, the culture medium was replaced with αMEM containing 10% FBS. Cultures between the 4th and 6th passages were used in this study.
THP-1 cell culture
The THP-1 cell line was obtained from the China Center for Type Culture Collection and cultured in RPMI 1640 medium (Gibco) containing 10% FBS. Before stimulation, THP-1 cells were differentiated into macrophages by incubation with 50 nM phorbol 12-myristate 13-acetate (PMA; #79346, Sigma‒Aldrich, St Louis, MO, USA) for 36 h.
Stimulation of canonical pyroptosis
As previously described, LPS priming plus nigericin stimulation was used to induce NLRP3 inflammasome-mediated canonical pyroptosis23. PDLFs, DPCs and THP-1 cells were primed with LPS (1 µg/ml, #tlrl-pglps, InvivoGen, San Diego, CA, USA) for 4 h and then stimulated with 20 µM nigericin (#N7143, Sigma‒Aldrich) for 2 h.
Poly(dA:dT) transfection was utilized to trigger AIM2 inflammasome-mediated canonical pyroptosis as described in a previous study24. In brief, PDLFs, DPCs and THP-1 cells were transfected with 2 µg/ml poly(dA:dT) (#tlrl-patn, InvivoGen) using Lipofectamine 2000 (#11668019, Invitrogen, Carlsbad, CA, USA). Two micrograms of poly(dA:dT) and 5 µl of Lipofectamine 2000 were diluted separately in 125 µl of Opti-MEM (Gibco) for 5 min. Then, the dilutions were mixed and incubated for 30 min at room temperature (RT). The mixture was added to cells in six-well plates with a final volume of 1 ml. The same proportions of poly(dA:dT) and Lipofectamine 2000 were used for assays in 96-well plates with final volumes of 100 µl.
Stimulation of noncanonical pyroptosis
As described in previous study, 2 µg/ml LPS from E. coli O111:B4 (#L4391, Sigma‒Aldrich) was transfected into PDLFs, DPCs and THP-1 cells using Lipofectamine 2000 to induce noncanonical pyroptosis20. The method for transfecting LPS was similar to that for transfecting poly(dA:dT). To investigate the inhibitory effects, cells were pretreated with DMF (#V900731, Sigma‒Aldrich) or Z-LEVD-FMK (#ALX-260-142-R100, Enzo Life Sciences, Farmingdale, NY, USA) for 1 h before LPS transfection in some experiments.
LDH release assay
Cell death was measured using a CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (#G1780, Promega, Madison, WI, USA). Cells were plated in 96-well plates and treated as indicated. Fifty microliters of supernatant in each well was transferred to a new plate and mixed with 50 µl of CytoTox 96 Reagent. After incubation at RT for 30 min, the absorbance was measured at 490 nm using a plate reader (BioTek Instruments Inc.). For the maximum LDH release positive control, 10X lysis solution was added 45 min before the addition of CytoTox 96 Reagent. The percentage of LDH release was calculated as follows: ((LDHsample – LDHmedium)/(LDHmaximum – LDHmedium))×100%.
Immunoblotting
Cells were cultured in six-well plates, stimulated as indicated and lysed using cell lysis buffer (#P0013, Beyotime, Shanghai, China) supplemented with PMSF (#ST506, Beyotime). Supernatant proteins were precipitated using acetone, centrifuged and resuspended in ddH2O containing 8 M urea. Then, the proteins were separated by SDS–PAGE, transferred to PVDF membranes (Sigma–Aldrich), and blocked with QuickBlock™ western blocking buffer (#P0252, Beyotime) for 15 min at RT. The membranes were incubated overnight at 4°C with the indicated antibodies: anti-caspase-1 (1:1000, #3866S, Cell Signaling Technology (CST), Boston, MA, USA), anti-GSDMD (1:200, #sc-81868, Santa Cruz, Dallas, TX, USA), anti-cleaved GSDMD (1:1000, #36425S, CST), anti-PARP (1:1000, #9542, CST), anti-IL-1 beta (1:1000, #ab216995, Abcam, Cambridge, MA, USA), anti-caspase-4 (1:1000, #ab238124, Abcam), anti-NLRP3 (1:1000, #ab214185, Abcam), anti-AIM2 (1:100, #sc-515514, Santa Cruz), anti-ASC (1:200, #sc-514414, Santa Cruz) and a HRP-conjugated β-actin rabbit mAb (1:5000, #AC028, ABclonal, Wuhan, China). After incubation with secondary antibodies, proteins were visualized using an Odyssey Imaging System (LI-COR).
CCK8 assay
The cytotoxicity of DMF was evaluated using a CCK8 assay kit (#CK04, Dojindo, Kumamoto, Japan). PDLFs and DPCs were plated in 96-well plates and treated with different concentrations of DMF. After the indicated times, the culture medium was replaced with 100 µl of fresh αMEM containing 10 µl of CCK-8 reagent. After incubation at 37°C for 1 h, the absorbance at 450 nm was measured. Cytotoxicity was determined as follows: ((Absorbancesample – Absorbancemedium)/(Absorbancecontrol – Absorbancemedium))×100%.
Cell viability assay
Cell viability was determined by measuring ATP levels using a commercial CellTiter-Glo Luminescent Cell Viability Assay kit (#G7570, Promega). In brief, cells were seeded in an opaque 96-well plate and treated as indicated. The plate was equilibrated to RT before measurement. Then, 100 µl of assay reagent was added to each well. The contents of each well were mixed for 2 min on an orbital shaker to induce cell lysis. After incubation at RT for 10 min, luminescence was measured using a plate reader (BioTek Instruments Inc.). Cell viability was calculated as follows: ((Luminescencesample – Luminescencemedium)/ (Luminescence control – Luminescence medium))×100%.
PI staining
Treated cells were stained with 2 µg/ml propidium iodide (PI; #ST511, Beyotime) for 15 min at RT. Then, the cells were fixed with 4% paraformaldehyde and observed under a fluorescence microscope (Olympus IX83).
Flow cytometry
Cell death was measured by flow cytometry. PDLFs and DPCs were plated in six-well plates and pretreated with the indicated concentrations of DMF 1 h before LPS transfection. Eight hours after transfection, cells were harvested using an EDTA-free trypsin solution (#BL527A, Biosharp, Guangzhou, China), centrifuged, resuspended in binding buffer and labeled with FITC-conjugated annexin V and PI using an apoptosis detection kit (#AP101, MultiSciences, Hangzhou, China). Finally, the labeled cells were analyzed with a flow cytometer (CytoFLEX, Beckman Coulter Inc.).
Confocal microscopy
Cells were seeded in 48-well plates containing coverslips and treated as indicated. After being washed with PBS, the cells were fixed with 4% paraformaldehyde. The fixed cells were permeabilized using 0.1% Triton, blocked with QuickBlock™ blocking buffer (#P0226, Beyotime) and incubated with an anti-cleaved N-terminal GSDMD antibody (1:200, #ab215203, Abcam) overnight at 4°C. Then, the cells were stained with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:100, #ANT024, AntGene, Wuhan, China) for 1 h at RT. The slices were mounted with fluorescent mounting medium containing DAPI (#ZLI-9557, ZSGB-BIO, Beijing, China). The stained slices were observed using a confocal microscope (FV1200, Olympus).
Statistical analysis
The values are presented as the means ± SDs in this study. The data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. P values less than 0.05 were considered statistically significant.