F 12 ham

Six- to eight-week-old male NOD scid gamma (NSG^TM; NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ) were used to implant PA137 PDX tumours. Tumour samples were cut into 3–4-mm pieces and immediately placed in F-12 HAM (Sigma-Aldrich) medium supplemented with 50% fetal calf serum and 50 units ml⁻¹ penicillin and 50 μg ml⁻¹ streptomycin. Tumour pieces were embedded into Matrigel before orthotopic implantation into mice. After implantation of tumour tissue, xenografts were allowed to grow for four weeks. When the tumour size reached 150 mm³, mice were randomly divided into two groups (vehicle control and pentamidine (10 mg kg⁻¹, every day, through intraperitoneal injection)) and assessed for survival. Tumour volume and body weight were recorded regularly during the treatment. The mice were euthanized when their tumours reached a size of 15 mm in diameter.

SH-SY5Y (European Collection of Cell Cultures, catalog number 94030304) cells were grown at 37°C in a 5% CO₂-humidified incubator on the culture medium composed of 1:1 Earle’s Balanced Salt Solution (EBSS, Sigma Aldrich) and F12 HAM (Sigma Aldrich) supplemented with 15% fetal bovine serum (FBS, Gibco), 1% Glutamine (Gln), 1% Non-Essential Amino Acids (NEAA, Sigma Aldrich), and 1% Penicilin-Streptomicin (P/S, Invitrogen). Petri dishes with confluent SH-SY5Y cells were washed twice with cold PBS, followed by solubilization in lysis buffer, composed of 20 mM HEPES, 2mM EGTA, 1mM DTT, 1mM sodium orthovanadate, 1% Triton X-100, 10% Glycerol, 2μM leupeptin, 400 μM PMSF, 50 μM β-glycerophosphate, 100 μg/ml trasylol, pH = 8.1.The cells were scrapped on ice for 10 minutes and incubated on ice for 30 minutes with periodic vortexing every 5 minutes, followed by centrifugation for 20 minutes at 14000 g at 4°C. The supernatant was saved for further use. Protein concentration was quantified using the BCA protein assay kit (Pierce).

A human retinal pigment epithelial cell line (ARPE-19) was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium-nutrient mixture F-12 HAM (Sigma) containing 10% fetal bovine serum and 1% penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) under a humidified atmosphere containing 5% CO₂ at 37°C. The cells were plated on 35 mm dishes at a density of 3 × 10⁴ cells/cm², grown to preconfluence, and starved of serum for 24 h before experiments were performed.
It has been reported that ARPE-19 cells became spindle-shaped and gathered together to form piled up cellular aggregates after culture for 48–72 h in the presence of both TNF-α and TGF-β₂, with these aggregates being termed EMT-associated fibrotic deposits [19 (link)]. We induced these cellular aggregates according to the method described by the authors, and utilized this phenomenon as an in vitro model of the EMT.

Human rhabdomyosarcoma (TE671) and lung cancer cell lines (A549, H2170 and H1299) were obtained from the American Type Culture Collection (ATCC). Human larynx cancer cells (RK33 and RK45) were derived from patients with diagnosed larynx squamous cell carcinomas. Cancer tissue was removed from the larynx after total laryngectomy and established as a stable cell line, as previously described [31 (link)]. Normal human primary fibroblast culture (HSF) was obtained by the outgrowth technique from skin explants of a young person, using a method routinely ongoing in our lab (Local Ethical Committee permission No KE0254/298/2015). TE671 cell line was maintained in nutrient mixture F-12 Ham culture medium (Sigma Chemicals, St. Louis, MO, USA). A549 was grown in a 1:1 mixture of DMEM and nutrient mixture F-12 Ham (Sigma Chemicals, St. Louis, MO, USA). RPMI 1640 medium (Sigma Chemicals, St. Louis, MO, USA) was used for culture of H2170, H1299, RK33, and RK45 cell lines and HSF. All culture media were supplemented with 10% fetal bovine serum (FBS) (Sigma Chemicals, St. Louis, MO, USA), penicillin (100 µg/mL) (Sigma Chemicals, St. Louis, MO, USA), and streptomycin (100 µg/mL) (Sigma Chemicals, St. Louis, MO, USA). Cultures were kept at 37 °C in a humidified atmosphere of 95% air and 5% CO₂.

Human urothelial carcinoma cell line RT4 (HTB-2, ATCC, Manassas, VA, USA) was grown in basal media consisting of equal parts of advanced Dulbecco’s Modified Eagle’s Medium (A-DMEM) (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (HAM) (Sigma-Aldrich, Merck, Darmstadt, Germany) supplemented with 5% fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA, USA) and 4 mM GlutaMAX (Gibco, Life technologies, Carlsbad, CA, USA) in 75 cm² culture flasks. For the experiments, RT4 cells were seeded on coverslips in 6-well plates (for fluorescence microscopy) or 96-well plates without coverslips (for measuring fluorescence intensity) at a seeding density of 1 × 105 cells/cm² and grown until reaching 80–90% confluence. To mimic a proinflammatory environment, cells were treated with 20 ng/mL human recombinant TNFα (Cayman Chemicals, Ann Arbor, MI, USA) in serum-free basal media for 24 h, as previously described [45 (link),46 (link),47 (link)]. Untreated cells grown in serum-free basal media served as controls.

Whole blood was collected into 4-mL lithium-heparin containing tubes (Greiner Bio-One, 454029). For the whole blood stimulation, whole blood was diluted 1:10 with Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (Sigma-Aldrich) supplemented with 10% foetal calf serum and 5% penicillin streptomycin. Of the diluted mixture, 500 μL was aliquoted to a 24-well plate. Each well was stimulated with 5 μL of the stimulant Escherichiacoli K12 lipopolysaccharide (LPS) which is a Toll-like receptor 4 (TLR4) agonist. Dysregulation of signalling pathways associated with TLR4 receptors is one of the most replicated findings in relation to alcohol and inflammatory processes.⁴⁰ (link) The blood was harvested after 24 h of incubation at 37 °C and stored at −80 °C for later analysis. The following cytokines were analysed both in unstimulated and TLR4 agonist-stimulated conditions: Tumour Necrosis Factor α (TNF-α), Interferon-γ (IFN-γ) and Interleukins (IL-1β, IL-2, IL-10, IL-6, IL-8). Cytokine levels were quantified using the V-PLEX Proinflammatory Panel 1 (human) Kit (MSD, K15049D). Cytokine quantification was done according to the manufacturer's guidelines with one modification, where a total of 100 μL (50 μL in duplicate) sample was added directly onto the plate without dilution (applied for both stimulated and unstimulated conditions).