S2228

J774A.1 cells, a mouse monocyte/macrophage cell line, were propagated in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂.
T. pyogenes (strain 0912) was cultured in Martin broth medium with 10% fetal bovine serum under aerobic conditions.
Primary antibodies included rabbit monoclonal anti-GSDMD (ab209845, Abcam), rat monoclonal anti-caspase-1(BL-645102, Biolegend), rabbit monoclonal anti-caspase-11 (ab180673, Abcam), mouse monoclonal anti-NLRP3(AG-20B-0014, Adipogen), and mouse anti-GAPDH antibody (GTX627408, GeneTex). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZB-2305, ZSGB-BIO), HRP-conjugated goat anti-rabbit IgG (ZB-2301, ZSGB-BIO) and HRP-conjugated goat anti-rat IgG secondary antibody (ZB-2307, ZSGB-BIO).
Chemicals included VX-765 (Capase-1 inhibitor) (S2228, Selleck), MCC950 (NLRP3 inhibitor) (S8930, Selleck), Necrostatin-1 (Nec-1) (receptor-interacting serine/threonine protein kinase 1 [RIP1] inhibitor) (A4213, APExBIO), Necrosulfonamide (NSA) (mixed lineage kinase domain-like protein [MLKL] and GSDMD inhibitor) (B7731, APExBIO), and Wedelolactone (Wed) (caspase-11 and NLRP3 inhibitor) (HY-N0551, MedChemExpress).

Human PDL progenitor cells were isolated as previously described and were identified following previous protocols,^{34 (link)} which used at passage 4. Compressive force loading was provided by glass layers and 50 mL plastic tube caps containing weighed metal balls as previously described.^{35 (link),36 (link)} 1.5 g/cm² compressive force was applied to PDL progenitor cells for different time points (3–24 h), and different compressive force (0.5–2.0 g/cm²) was applied to PDL progenitor cells for 6 h. In addition, after being subjected to 1.0 g/cm² and 1.5 g/cm² compressive force for 6 h, PDL progenitor cells were collected for further experiments of optical microscope (OM, Olympus, Japan), scanning electron microscope (SEM) and transmission electron microscope (TEM).
To confirm the influence of pyroptosis under mechanical stimuli, pyroptosis activator PPVI (4 μmol/L), pyroptosis inhibitor MCC950 (10 μmol/L) and Caspase-1 inhibitor Belnacasan (VX765, 20 μM, S2228, Selleck) were added to PDL progenitor cells for 18 h in advance, then 1.5 g/cm² force was applied to PDL progenitor cells for 6 h.^{31 ,37} In addition, TRPV4 inhibitor GSK219 (10 mmol/L, Selleck) were applied to PDL progenitor cells for 1 h and then stimulated with force loading (1.5 g/cm², 6 h).¹⁷ PDL progenitor cells without force-loaded and drug treatment served as controls.