Bzatp

Peripheral blood mononuclear cells were suspended at the concentration of 5×10⁵ cells/ml of 10% FBS supplemented RPMI (Gibco, Thermo Scientific, Waltham, MA, United States) without antibiotics and placed into 12-well plates for cell culture, using four wells for each patient. After an overnight incubation at 37°, 5% CO₂, non-adherent cells were removed and the adherent mononuclear cells (90% macrophages, as verified detecting shape modification at optical microscopy) were incubated in 10% FBS-supplemented RPMI under the following conditions: (1) no additions (control) for 5 h; (2) 1 μg/ml lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, United States) for 4 h; (3) 1 μg/ml LPS for 4 h, followed by 300 μM BzATP (Sigma-Aldrich) for 1 h; (4) none for 4 h, followed by 300 μM BzATP for 1 h. At the end, the supernatants were collected, centrifuged and frozen at -80°C until use.

1x10⁶ cells were either mock- or MVA (MVA-PK1L-OVA) infected at an MOI of 1 for either 4h or 20h. For Ca²⁺ measurements infected cells were placed in a falcon for loading with 4µM FURA-2 AM (Sigma-Aldrich) in saline solution (12.5mM NaCl, 0.5mM KCl, 0.1mM MgSO₄, 2mM HEPES, 0.55mM D-glucose, 0.5mM NaHCO₃ (all Sigma-Aldrich)) supplemented with 0.5mM CaCl₂ (pH 7.4, Merck) and 250µM sulfinpyrazone (Sigma-Aldrich) at 37°C for 20min. Cells were then washed, resuspended in saline solution and stimulated with the indicated concentrations of Bz-ATP (Sigma-Aldrich) and 1µM ionomycin (Invitrogen) for the recording of intracellular Ca²⁺ release. For detection of pore opening at the cell membrane, cells were loaded with 2µL ethidium bromide (EtBr (Sigma-Aldrich)) and stimulated with 200µM Bz-ATP and 100µM digitonin (Sigma-Aldrich). Measurements were done in a thermostat quartz cuvette using a Perkin-Elmer KS50 rotating and heating system at a wavelength of 340/380nm (excitation) and 505nm (emission) for intracellular Ca²⁺ release and at a wavelength of 360nm (excitation) and 580nm (emission) for EtBr- pore opening assay.

HEPES, ATP, bzATP, NaCl, KCl, CaCl₂, MgCl₂ and glucose were obtained from MERCK (Darmstadt, Germany). Penicillin (10000U) and streptomycin (10 mg/mL) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA), whereas Trypsin 10X, Hank’s solution, ATP determination kit, Fura-2AM, Dulbecco’s Modified Eagle Medium (DMEM), Phosphate-Buffered Saline (PBS) and Etd bromide (10 mg/mL) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (ab273068, Wuhan-Hu-1 variant: MN908947) and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Abcam (Cambridge, UK). The mimetic peptides gap19 (KQIEIKKFK, intracellular loop domain of Cx43), TAT-L2 (YGRKKRRQRRR-DGANVDMHLKQIEIKKFKYGIEEHGK, intracellular loop domain of Cx43) and ¹⁰panx1 (WRQAAFVDSY, first extracellular loop domain of Panx1) were obtained from Genscript (New Jersey, USA).