Crystal violet assay

QH and OE33 cells were seeded at 15,000 cells per well in a 96-well cell culture plate, reverse transfected as described above and allowed to incubate for 24 h. Cell supernatant was removed and the cells were washed with 100 μL PBS/Mg²⁺ buffer (130 mM NaCl, 5 mM KCl, 1 mM Na²PO₄, 1 mM CaCl₂, 1 mM MgCl₂ and 25 mM HEPES, pH 7.4) and subsequently incubated for 30 min at 37 °C in PBS/Mg²⁺ containing 5 µM 2′,7′-dichlorofluorescein (Invitrogen), 0.3 µM mitotracker (Invitrogen, California, USA) or 5 µM rhodamine 123 (Sigma, St. Louis, MO, USA) to assess reactive oxygen species production, mitochondrial mass and mitochondrial membrane potential respectively. 2′,7′-dichlorofluorescein and mitotracker were solubilized in DMSO, whereas rhodamine 123 was solubilized in ethanol. Cells were washed with 200 μL PBS/Mg²⁺ buffer, the buffer discarded, 100 μL fresh buffer added and fluorescence read with excitation and emission wavelengths of 485 nm and 538 nm respectively (Thermo Scientific, Fluoroskan Ascent FL, Waltham, MA, USA). All measurements were normalized to cell number using the crystal violet assay (Sigma) (9).

The human breast adenocarcinoma cell line MCF-7 (ATCC® HTB­22TM) was purchased from the American Type Culture Collections. Cells were cultured in appropriate medium (Sigma-Aldrich, MO, USA) as per the ATCC protocol with the addition of 10% FBS (Sigma-Aldrich, MO, USA).
Cell viability was determined by Crystal Violet Assay (Sigma-Aldrich, MO, USA).

The optical density was measured using a UV–VIS spectrophotometer (model DU-730; Beckman Coulter Inc., Fullerton, CA, USA). The metabolite analysis in the supernatants was carried out by high-performance liquid chromatography (HPLC) using a Waters 2414 refractive index detector (Waters Corp, Waltham, MA, USA) equipped with a Shodex SH1011 column (Shodex, Tokyo, Japan). The column temperature was 75 °C, and 10 mM sulfuric acid was used for the mobile phase at a flow rate of 0.6 mL/min. The biofilm was detected using a Crystal Violet assay (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s protocol [23 (link)] and also analyzed by scanning electron microscopy (SEM, Hitachi S-4700, Tokyo, Japan).

PDAC cells were seeded on 96-well plates (9 × 10³ cells/well). Forty-eight hours later, cell growth was measured by Crystal Violet assay (Sigma, Milan, Italy) according to the manufacturer’s protocol, and the absorbance was measured by spectrophotometric analysis (A595 nm).

AsPC-1 cells were seeded in 96-well plates (8 × 10³ cells/well). Forty-eight hours later, cell growth was measured by Crystal Violet assay (Sigma, Milan, Italy) according to the manufacturer’s protocol, and the absorbance was measured by spectrophotometric analysis (A₅₉₅ nm).

The cell viability was assessed using the crystal violet assay (Sigma-Aldrich Co., St. Louis, MO, USA). Briefly, 2 × 10⁴ cells per well were seeded into a 24-well plate and incubated overnight. Following that, cells were treated with various concentrations of bortezomib (0–100 nM) and PCC (0–2000 nM), and the plates were incubated for 48 h. After incubation, cells were rinsed with PBS and fixed with 10% formalin for 15 min. After washing, the cells were stained with 0.1% crystal violet and incubated for 30 min at room temperature. The excess stain was removed by washing the plates twice and air-drying. Then, the cell-bound crystal violet was suspended in 10% acetic acid, and the absorbance (A) was read at 570 nm using a BioTek Synergy H1 multimode plate reader (BioTek Instruments, Inc., Winooski, VE, USA).

Cells were seeded in 96-well plates and the day after were incubated with various compounds at the indicated conditions or transfected with the indicated constructs (see figure legends). At the end of the treatments, cell growth was measured by Crystal Violet assay (Sigma-Aldrich) according to the manufacturer’s protocol, and absorbance was measured by spectrophotometric analysis (A 595 nm).