Cytochalasin b

To assess the primary mechanism for MRSA killing by PMNs from healthy donors, phagocytosis of PMNs was inhibited by 10 µM cytochalasin-B (MedChem Express, cat#14930-96-2) and NETs released from PMNs were degraded by 10 µg/mL DNAse I (Sigma-Aldrich, St. Louis, MO, USA, cat#AMPD1-1KT). After PMN isolation from the blood of healthy donors, PMNs were either untreated, pretreated with 10 μM cytochalasin-B, or 10 μg/mL DNAse I for 20 min at 37 °C. Following pretreatment, PMNs were infected with opsonized MRSA isolates as described above. The control samples were added with bacteria and drug alone (no PMNs), to assess whether drug affects bacterial growth itself.

mDPC6T cells stimulated with LPS and ATP were utilized to simulate the pyroptosis during the pulpitis pathological process. In detail, mDPC6T cells were first primed with 1 μg/mL LPS (#LPS25, Sigma Aldrich) for 24 h, followed by treatment with 2 mM ATP (#HY‐B2176, MedChemExpress) for 24 h (referred to as LA‐mDPC6T). To inhibit mitochondrial OS, mDPC6T cells were pre‐treated with 5 μM MitoTEMPO (#HY‐112879, MedChemExpress) or 2 μM NAC (#HY‐B0215, MedChemExpress) for 4 h prior to stimulation with LPS and ATP. To inhibit mitochondrial transfer and tunnelling nanotube (TNT) formation, cells were pre‐treated with 1 μM cytochalasin B (HY‐16928, MedChemExpress) for 6 h to interfere polymerization and interaction of actin. For signalling pathway inhibition, cells were pre‐treated with 1 μM NF‐κB inhibitor Bay 11‐7082 (#HY‐13453, MedChemExpress) or 5 μM IKKβ inhibitor ML120B (#HY‐15473, MedChemExpress) for 4 h before experiments. To generate the mBMSCs without the mitochondrial function (ρ^omBMSCs), cells were cultured in a medium additioned with 1 μg/mL ethidium bromide (15585011, Invitrogen), 100 μg/mL pyruvic acid (HY‐Y0781, MedChemExpress) and 50 μg/mL uridine (HY‐B1449, MedChemExpress) for at least 1 month. For the TNF‐α treatment, the mBMSCs were treated with different concentrations of mouse TNF‐α (#HY‐P7090, MedChemExpress) for 24 h.

PD98059, XAV-939, wortmannin, 6-bromoindirubin-3′-oxime (BIO), and Cytochalasin B were purchased from MedChemExpress (NJ, USA). The chemical reagents were dissolved in dimethyl sulfoxide at a concentration of 10 mM, 10 mM, 1 mM, 10 mM, and 10 mM, respectively. And the final concentration of the chemical reagents in the culture medium was: 20 μM, 1 μM, 25 nM, 2 μM, and 20 μM, respectively.