Fluorodish plate

For biofilm generation, samples were prepared in FluoroDish plates (World Precision Instrument Inc., China) that contained 3 mL of sterile tripticase soy broth supplied with sucrose, 1%. The plates were prepared with 10 μL of bacterial inoculum (5 × 10⁵ UFC·mL⁻¹) for incubation at 5% of atmosphere CO₂, at 37°C for 48 hours. Biofilm was generated in a liquid medium, and different concentrations of EP were added (0.1 to 1.96 μg·mL⁻¹).

Cells were planted on fluorodish plates (35 mm, World Precision Instruments, Sarasota, FL, USA) for experiments. After OGD/R for 24 h, cells were washed with HBSS (calcium-free) and loaded with 5 μM Fluo-3 AM (Beyotime Institute of Biotechnology) in HBSS (calcium-free) for 60 min at 37 °C. The cells were then washed with HBSS (calcium-free) and incubated in HBSS (calcium-free) for an additional 30 min. Images were captured by an inverted confocal microscope (Live5; Carl Zeiss, Inc., Tokyo, Japan) to detect the fluorescence intensity of cells, which represented the calcium concentration in the cytoplasm. It took 90 seconds to determine basal fluorescence intensity, then 0.2 μM Tg or 2 μM of ATP were added directly to the cell solution to trigger ER Ca²⁺ depletion⁴⁹ or IP3R- and IP3-induced Ca²⁺ release (IICR)^{50 (link)}; cells were then continuously observed for 6 min. Cells were excited by a 488-nm laser, and images were acquired at 5 s intervals in time-lapse mode and subsequently analyzed using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). These data were quantified as the area under the curve (AUC) for all peaks.

PrecIL-1α fused to GFP was cloned into the pEGFP-N1 vector (Clontech) as was previously described by Cohen et al.5 (link). The substitute mimicry mutations of IL-1α-K82 was performed using the site-directed mutagenesis PCR reaction with the forward primer 5′-phospho-TCAAGCAACGGGCAGATTCTGAAGA or 5′-phospho-TCAAGCAACGGGCGGATTCTGAAGA together with reverse primer 5′-phospho-CGTTGCTGATACTGTCACCCGG with the precIL-1α-GFP encoding vector as a template and the Phusion DNA polymerase enzyme (New England Biolabs), according to the manufacturer’s protocol. For transient transfections, 5 × 10⁴ B16 mouse melanoma cells were cultured in 35 mm Fluorodish plates (World Precision Instruments) for 24 h and then transfected with the various IL-1α plasmids using the JetPEI transfection reagent (Polyplus Transfection). 16 h after transfection, the GFP signal was visualized with the Olympus Fluoview FV1000 confocal microscope (Olympus) using 405 nm and 488 nm laser lines. Nuclear counter-staining of the transfected cells was performed with Hoechst 33342 (Fluka).