C6158

Plaque assays to measure infectious virus counts were performed as described before [27 (link)]. Briefly, Vero E6 cells were seeded in 6-well cell culture dishes to reach complete confluency the next day. Cells were washed once with 2 mL warm PBS and incubated with dilutions of cell culture supernatants in 200 μL complete DMEM for 1 h at 37 °C. The virus inoculum was then removed, and cells were overlaid with DMEM containing 2% FBS and 0.8% agarose (#A6013, Sigma-Aldrich, St. Louis, MO, USA). After 72 h incubation, cells were fixed with 4% formalin, and plaques were visualized via crystal violet staining (#C6158, Sigma-Aldrich, St. Louis, MO, USA).

The soft agar assay was performed as previously described (19 (link)). The base layer of each well consisted of 1.5 mL of serum-free 2X DMEM (Welgene, LM 201-50) with a final concentration of 0.5% Noble agar (BD Biosciences). After bottom agar solidification, 1.5 mL of 0.35% agar containing NIH3T3 cells (20,000) was seeded on the bottom agar layer and incubated for 14 days. Medium was changed every 2 days for 2 weeks. Colonies were fixed with 4% paraformaldehyde and then stained with 0.05% crystal violet (Sigma-Aldrich, C6158).

HUVEC were detached, washed twice in PBS and re-suspended in EBM-2 medium with 10% FBS. 5.10⁴ cells were seeded in the upper chamber of a Transwell insert (PTFE membrane with 8 μm diameter pores, Corning). The lower chamber was filled with 1 mL of EBM-2 with 20% FBS with or without DEET. After 24 h, migrated cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were rinsed two times with washing buffer, stained with crystal violet (1 mg.mL⁻¹ in 2% of ethanol, Sigma–Aldrich, C6158) for 10 min at room temperature and extensively washed with distilled water. Then, SDS 2% was added and incubated for 30 min at room temperature. Absorbance was then evaluated using a Mithras LB940 multimode microplate reader at 550 nm (Berthold Technologies).

Cell numbers were determined after crystal violet staining (Sigma‒Aldrich, C6158) [23 (link)]. Cells were washed with phosphate-buffered saline (PBS), fixed and stained for 20 min with 0.2% crystal violet in 2% ethanol. In order to eliminate dye precipitates, the staining solution was filtered prior its use with a syringe-driven filter unit (0.22 µm pore size, Millipore, SLGP033RS). Cells were then washed with water to remove excess dye. Once dried, the dye was dissolved in 10% acetic acid. Cell number was determined by absorbance measurement at 595 nm with the multilabel plate reader VICTOR™ X3 (PerkinElmer, Courtaboeuf, France).

For the growth curves: A U-bottomed, clear 96-well plate (Greiner Bio-One #650161) was prepared with either LB or LB supplemented with 30 mM itaconate. Each well was inoculated with 1.5 × 106 bacteria. Absorbance at 600 nm was read every 30 minutes for 9 or 18 hours on the SpectraMax M2 plate reader, as the plate incubated at 37 °C with shaking.
For the growth and biofilm assay: A flat-bottomed, clear, 96-well plate was prepared with LB or LB with 0.5% glucose (w/v), supplemented with serially diluted, pH-corrected itaconate. Each well was inoculated with 1.5 × 106 bacteria, and the plate was left to incubate statically overnight at 37 °C. The next morning, absorbance at 600 nm was determined on an Infinite M200 plate reader (Tecan). To stain the biofilm, the supernatant was discarded, the plate was washed and dried, and the biofilm was fixed with 100% methanol, then stained with 1% crystal violet (w/v, Sigma #C6158). After discarding the staining solution and washing and drying the plate, the stained biofilm was resuspended in 33% acetic acid (v/v, Acros Organics #222140010). Absorbance at 540 nm was determined on the Infinite M200 plate reader using iControl v1.10.4.

Cells (500 per well) were seeded in a 6-well plate and cultured for 14 days. After 14 days of culture, 4% paraformaldehyde and 0.5% crystal violet (C6158, Sigma-Aldrich, St. Louis, USA) were used to fix and stain the cells, respectively. The colonies were counted under a light microscope.

Cell viability was analyzed 48 h and 72 h after TKI treatment by crystal violet staining (C6158, Sigma, France), as previously described [42 (link)]. Plated cells were washed with 1× PBS and incubated min with 0.1% crystal violet solution for 20 min. After several washes, 10% acetic acid solution was added to lyse-stained cells. Absorbance was measured at 595 nm using an Infinite 200 Pro Microplate Luminometer (Tecan Trading AG).