Six well plate

T98G glioma cell line was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were cultured in 25-ml flasks in medium composed of Advanced MEM supplemented with 10% fetal bovine serum, 2 mM glutamine and a penicillin–streptomycin solution, in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. For subcultures, cells were harvested every third day in trypsin-EDTA (0.25% trypsin, 1 mM EDTA) solution. For the gene expression assay, T98G cells were plated onto six-well plates (Nunc) at a density of 3 × 10⁵ cells per well. For the cell viability assay, the cells were seeded onto 96-well plates, at the density of 5 × 10⁴ cells per well. The following substances were used: fetal bovine serum, penicillin–streptomycin solution (5000 units/ml penicillin and 5000 g/ml streptomycin sulphate in normal saline), phosphate buffered saline (PBS; pH 7.4) and trypsin–EDTA were purchased from Invitrogen (Carlsbad, CA, USA). Advanced MEM was obtained from Gibco (Paisley, Scotland, UK).

Polysaccharide intercellular adhesion (PIA) in the biofilms of the ΔphoU1 and ΔphoU2 mutants was semiquantified by a dot blot assay with a wheat germ agglutinin (WGA)-horseradish peroxidase (HRP) conjugate as described previously (63 (link), 64 (link)). Briefly, overnight cultures of the S. epidermidis strains were inoculated into six-well plates (Nunc) and incubated at 37°C for 24 h. Biofilms were scraped off the bottoms of the wells, resuspended in 0.5 M EDTA, and centrifuged (13,000 × g, 5 min) after they were heated at 100°C for 5 min. The supernatant was treated with proteinase K (20 mg/ml) at 37°C for 3 h and inactivated at 100°C for 5 min. Serial dilutions of the PIA assay extract were transferred to a nitrocellulose membrane (Millipore, Billerica, MA) using a 96-well dot blot device (Biometra GmbH, Gottingen, Germany). The air-dried membrane was blocked with 5% (wt/vol) skim milk and subsequently incubated with the WGA (3.2 μg/ml)-HRP conjugate for 1 h (Lectinotest Laboratory, Lviv, Ukraine). HRP activity was visualized by chromogenic detection using 4-chloride-1-naphthol (Sigma) as the substrate. Quantitation of PIA was represented as the highest dilution of the supernatant in which HRP was detectable.

The HT29 cell line was obtained from LONZA (Basel, Switzerland). NCM460 cells were from INCELL Corporation (San Antonio, TX, USA). SW480 cells were a kind gift from Prof. Alberto Muñoz (CSIC, Madrid, Spain). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and fetal bovine serum (FBS) were sourced from Lonza (Basel, Switzerland). L-glutamine was from Gibco (Barcelona, Spain). Trypsin-EDTA was from LONZA (Verbiers, Belgium). Poly-L-Lysin was from Marlenfeld GmbH (Lauda-Könlgshofen, Germany). Six-well plates were from NUNC (Thermo Scientific, Waltham, MA, USA). Dishes 10 cm² in diameter were from Corning (NY, USA). DFMO was from TOCRIS (Bristol, UK). Fura2/AM and qPCR primers are from Invitrogen (Eugene, OR, USA). Cyclopiazonic acid (CPA) was from Sigma-Aldrich (Steinheim, Alemania). Antibodies against MCU and β actin were from Sigma (Madrid, Spain). The RNA extraction kit was a GeneMATRIX Universal RNA Purification Kit from EURx (Gdansk, Poland). Clariom D human microarrays (Affymetrix) were supplied by CABIMER (Andalucía, Spain). RNA-seq (Illumina) was provided by Sistemas Genómicos S.L (Valencia, Spain). PolyamineRED was from Funakoshi Co., Ltd., Tokyo, Japan). All other reagents were obtained from Sigma and Merck.

Human rhabdomyosarcoma and larynx cancer cells were placed on six-well plates (Nunc) at a density of 1 × 10⁵/mL. The next day, the medium was replaced with fresh medium containing different concentrations of imperatorin (10, 25, 50, and 100 µM) for 48 h. After that, cells were harvested and washed twice with PBS. Next, cells were fixed and permeabilized using the cytofix/cytoperm solution according to the manufacturer’s instructions of Phycoerythrin (PE) Active Caspase-3 Apoptosis Kit (BD Pharmingen). Finally, cells were washed twice in the perm/wash buffer prior to intracellular staining with PE-conjugated anti-active caspase-3 monoclonal rabbit antibodies. Labelled cells were analyzed by flow cytometer FACSCalibur (Becton Dickinson, San Jose, CA, USA), operating with CellQuest software to quantitatively assess the caspase-3 activity.

ES-E14TG2a mouse embryonic stem cells, P19 mouse embryonic carcinoma cells, and porcine embryo fibroblast cells were plated into six-well plates (Nunc) (Figure 2). Each reporter construct was transfected into cells using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's protocol. Cell lysate was prepared 48 h after transfection. Luciferase activity was detected using a Luciferase Assay System (Promega). Transfection of the pGL3-basic vector (Promega), which does not contain inserted DNA, was used as a control to determine the background level of luciferase activity.

AVPR1A, CXCR4, and ACKR3 siRNA gene silencing was performed as described previously [22 (link), 24 (link), 25 (link), 30 (link)]. In brief, hVSMCs were grown in 2 ml Accell siRNA delivery media per well (Dharmacon) in six-well plates (Nunc). Commercially available Accell AVPR1A, CXCR4, or ACKR3 siRNA was reconstituted with 1× siRNA buffer to a stock concentration of 100 μM. Cells were then transfected with 1 μM siRNA and incubated for 72 h at 37°C, 5% CO2. Accell NT-siRNA pool was used as a negative control. After 72 h, cells were assayed for receptor cell surface expression