Slide 8 well slides

Overnight cultures of S. parasanguinis (mCherry), S. mutans (GFP), or C. albicans were subcultured and grown to an optical density of 0.5 at an absorbance of 600nm. All biofilms were grown in TSBYE + 1% sucrose with or without 2mM nitrite in µ-Slide 8 well slides (Ibidi, Gräfelfing, Germany, Cat #: 80826) and all inocula were seeded at 1x10⁴ CFU/mL. Biofilms were allowed to grow for 16 hours at 37°C with 5% CO₂. All biofilm wells were washed with PBS and wells with C. albicans were stained with calcofluor white for 15 minutes before imaging. A Nikon A1 + confocal laser scanning microscope (CLSM) (Nikon Instruments Inc., Melville, NY, USA) was used to image biofilms at 60x magnification and 3D images were acquired using the Nis Elements 5.0 Imaging Software (Nikon Instruments Inc., Melville, NY, USA). To enumerate colony forming units, all biofilms were gently washed with sterile PBS twice before adding 200 µL of sterile PBS for plating. The biofilms were scraped up with a 200 µL tip, vortexed for 10 s, and serially diluted. All dilutions were plated on Todd-Hewitt Broth or blood agar plates and incubated at 37 °C with 5% CO₂ for a minimum of 16 h before counting.

Cells were treated with 50 mM etomoxir and/or 200 nM INK128 for 48 h as described in Results. Cells were trypsinized and plated on Matrigel (Sigma-Aldrich, CLS356237) pre-coated Ibidi µ-Slide 8 Well slides (Ibidi, 80821). After 30 min of attachment, cells were stained for 1 h with SPY650-DNA (SpiroChrome, SC501) in culture medium with inhibitors at 37 °C, washed twice with HEPES-buffered saline (Sigma-Aldrich, 40010) and stained for 2 h with 20 µM FAOBlue (DiagnoCine, FNK-FDV-0033) in DMEM with inhibitors at 37 °C in 20% O₂ and 5% CO₂. Cells were washed once with HEPES-buffered saline and imaged with 200 µl HEPES-buffered saline. Imaging was done on a Zeiss Plan-Apochromat 20×/0.8 objective on the Zeiss LSM880 Airy microscope using longitudinal-section magnetic mode. Images were processed using Fiji ImageJ2 (version 2.3.0) and quantified using CellProfiler (version 4.2.1).