Crystal violet

CAL27 cells transduced with inducible shADAR1 (or shGFP) were seeded at 30,000 cells per well in a 12‐well plate. The following day, doxycycline was added at a final concentration of 1 µg/ml and cells were transfected with siLGP2 or siCtrl as described above. Doxycycline‐containing medium was refreshed 72 h post‐transfection. Cell confluency was measured every 4 h on a IncuCyte S3 Live‐Cell Analysis machine (Essen Bioscience) or at an end point of 120 h post‐siRNA transfection by Crystal Violet staining. For staining, cells were fixed in 100% ice‐cold methanol for 10 min, washed with PBS, and stained for 10 min with 0.1% Crystal Violet (V5265, Sigma) in 40% methanol. Stained plates were washed with water and dried, before imaging on a GelCount machine (Oxford Optronics Ltd.). Upon imaging, Crystal Violet was extracted from the stained cells by incubation in 15% acetic acid at RT for 20 min. The amount of extracted Crystal Violet was quantified by measuring the optical density at 590 nm using an Infinite M Plex plate reader (Tecan).

From 3.000 to 15.000 cells were seeded in 12-well plates, with three or six replicates per condition. Cells were maintained in culture medium for up to 8 days, with collection points every two days. Day 0 was defined less than 24 h after seeding. In the case of the catalytic dead mutant proliferation rescue, 0.1 µg/mL of doxycycline was added to the cells during curve seeding. For collection, cells were washed twice with PBS and fixed with 4% paraformaldehyde (Panreac). Staining was performed with 0.1% crystal violet (Sigma) in 10% methanol for 1 h, after which the wells were thoroughly washed with dH₂O and dried overnight at room temperature. crystal violet was dissolved in 10% acetic acid for 40 min, and cell density was quantified by measuring the absorbance at 595 nm using a Plate Infinite reader 200 Pro (TECAN). All data were normalised to day 0 measurements.

To evaluate if prodigiosin can inhibit bacterial biofilm formation, the biofilm assay was performed [35 (link)]. Briefly, overnight bacterial cultures were normalised to OD₆₀₀ = 0.4 (10⁸ CFU/mL) using their respective broth. The diluted bacterial cultures were then treated with 500 μg/μL of prodigiosin and added into 96-well microtiter plates (Greiner Bio-One, Austria) and incubated at 37°C for 48 hours. For each bacterial sample, the negative control used was their respective broth while the positive control used was S. aureus, a known high biofilm former [36 (link)]. After the 48-hour incubation period, each well was washed three times with 200 μL of 1× PBS after which 200 μL of 99% methanol was added into each well and left for 15 minutes at room temperature. Methanol was then discarded and the plate was allowed to dry for 15 minutes. Then, 200 μL of 0.5% crystal violet (Sigma-Aldrich, USA) was added into each well and left at room temperature for 5 minutes to stain the bacterial biofilm. Excess crystal violet was then washed away using distilled water and plates were left to dry completely at room temperature. Finally, 95% ethanol was used to dissolve the crystal violet bound to the biofilm. Optical readings of the dissolved crystal violet at 570 nm were measured on the Magellan^TM software using the Sunrise absorbance microplate reader (Tecan, Switzerland).

Overnight cultures in BHILC medium were diluted to an OD₆₀₀ of 0.05 and transferred to three Greiner Bio-one polystyrene flat-bottom 96-well plates, adding 150 μl per well. After 24 h of static incubation, one of the three plates was resuspended by pipetting to measure OD₆₀₀ using a Tecan Infinite-M200-Pro spectrophotometer. The two other plates were used for coloration, as follows. Cultures were removed by carefully pipetting the supernatant out and biofilms fixed with 150 μl Bouin solution (HT10132; Sigma-Aldrich) for 15 min. Bouin solution was removed by inversion, and the biofilms were washed once in water. The biofilms were stained with 150 μl of 1% crystal violet (V5265; Sigma-Aldrich) for 15 min without shaking and then washed in water twice and left to dry. All washes were made by flicking the plate. After drying the plate, crystal violet was dissolved with 200 μl absolute ethanol and transferred to a clean 96-well plate for OD₆₂₀ measurement (Tecan Infinite-M200-Pro spectrophotometer).

Biofilm formation was assayed essentially as described [29 (link)] with the following modifications. Overnight bacterial cultures were adjusted to a concentration of 1x10⁸ colony-forming units (CFU)/mL and then inoculated into 96-well plates with or without 50 μM epinephrine. The plates were then incubated statically at 37°C for 72 h. After incubation, non-adherent cells were removed by washing twice in phosphate-buffered saline (PBS; Sigma, St. Louis, MO). The biofilm was then fixed using 99% (v/v) methanol for 15 minutes and stained with 0.1% (w/v) crystal violet (Merck, New Jersey, USA) for 15 minutes. Finally, crystal violet was solubilized using 33% (v/v) glacial acetic acid (VWR international, Pennsylvania, USA). Optical density of the solubilized crystal violet suspension was then measured at 550 nm using a microplate reader (TECAN, Switzerland). Assays were performed with eight technical replicates on three separate occasions.

To study the cellular proliferation, 1 × 10⁵ cells were seeded in tetraplicates in a 6-well plate. Cell number was counted manually with a hemocytometer at three different time points after seeding for 24, 48, and 72 h. For the colony formation assay, 1000 cells per well were seeded in a 6-well plate in triplicates. Cell were also treated with cisplatin, LY294002, or a combination of cisplatin and LY294002 for 48 h before seeding and incubated at 37 °C in a 5% CO₂ incubator. After 14 days, cells were rinsed with 1X PBS, fixed in 5% glutaraldehyde for 20 min, and stained with 0.5% crystal violet (Sigma Aldrich, St. Louis, MO, USA) for 20 min. Plates were washed with water and dried before scanning. crystal violet was solubilized with 10% acetic acid and absorbance was measured at 450 nm in a microplate reader (Tecan, Mannedorf, Switzerland).