Dulbecco modified eagle medium (dmem)

Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC cell lines (American Type Culture Collection - Manassas, VA – United States) and cultured in growth medium (Dulbecco’s modified eagle’s medium – DMEM, Vitrocell, 00025) supplemented with fetal bovine serum (FBS) 10% from Gibco (12657029, South America) and used at passages 4–6. After serum deprivation in culture medium for 12 h, confluent HUVECs were treated with dextrin (10 mM) for 60 min or not (control) in the presence of BH₄ (100 μM, for 30 min), L-arginine (1 mM, for 30 min), or BH₄ plus L-arginine. Then, the cultured cells were lysed on ice in RIPA buffer supplemented with a cocktail of protease and phosphatase inhibitors, and the lysates were placed under non-denaturing conditions. The samples were prepared with 4x Laemmli sample buffer (Biorad, 1610747) plus betamercaptoethanol 10% (Biorad, 1610710) and were loaded on polyacrylamide gel 8%. During electrophoresis and protein transfer to the nitrocellulose membrane, the buffers were placed in an ice-water bath, and the whole apparatus was kept at 4 °C. The eNOS monomer and dimer forms were incubated with antibody against eNOS (1:1000, BD Bioscience, 610296) and α-tubulin or GAPDH were used to normalize the results. The bands were detected by a chemiluminescence substrate (Santa Cruz, SC-2048).

The samples were prepared as mentioned above in a mold with 5 mm diameter and 3 mm height and were sterilized with ultraviolet (UV) light for 30 min to prevent contamination. All the prepared samples for the MTT assay were incubated at 37 °C for 24 h immediately after mixing. According to ISO 10993-12 standards for preparing the release of fluids from solid form materials, the discs were immersed into 15 ml of Dulbecco’s modified Eagle medium (DMEM) (Vitrocell Embriolife, Campinas, SP, Brazil) and incubated for 24 h at 37 °C in a humidified 5% CO₂ environment. The extract material solutions were passed twice through the 0.22 μm filter to remove small particles, and the material extracts were obtained for the cytotoxicity tests according to ISO 19993-5 [23 (link)].

HEK293 cell line was cultured in DMEM (Vitrocell, São Paulo, Brazil) containing 10 μg·mL⁻¹ of streptomycin (Vitrocell) and supplemented with 10% fetal bovine serum (Vitrocell). HEK293_TOP cells which overexpress TOP protein were previously generated 42 and cultured under the same conditions.

MDCK cells (Madin-Darby Canine Kidney, NBL2; American Type Culture Collection-ATCC, Manassas, VA, USA) were acquired and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Vitrocell, Campinas, Brazil) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO) and maintained at 37 °C with 5% CO₂.
To analyze the effect of TGF-β1 on IDO expression, MDCK cells were seeded in 24-well plates (3X10⁴ cells per well). The cells were incubated with 1 ng/ml of TGF-β1 (R&D Systems Inc., Minneapolis, MN) in DMEM 1% FBS for 48 h. MDCK in DMEM 1% FBS without TGF-β1 was used as control. To promote IDO inhibition, we used DMEM containing 1 mM 1-methyl-D-tryptophan (MT; cat 452,483, Sigma-Aldrich, St. Louis, MO). All experiments were performed in triplicate.

The LLC cells (murine Lewis Lung Carcinoma, ATCC) were cultured under standard conditions at 37 ${}^{\circ }$ C and CO ${}_2$ 5% atmosphere. DMEM (Vitrocell) cell culture medium was supplemented with fetal bovine serum (FBS) 10% (v/v) (Sigma-Aldrich) and 4.5 mg/L glucose (Sigma-Aldrich). Cells were passaged regularly with a trypsin-EDTA solution (0.25% trypsin and 1mM EDTA).

Acquisition of T. cruzi trypomastigotes was by infection of LLCMK2 cells using trypomastigotes in T-25/75cm² containers at 37 °C with 5% CO₂ atmosphere in DMEM (Vitrocell, São Paulo, Brazil) and supplementation of 1% antibiotics and 2% SFB for 24 h. Then the trypomastigotes were plated and treated with p-coumaric acid derivatives. Cell viability was counted after 24 h of incubation at 37 °C. LC₅₀ was obtained [64 (link)]. While relative cell viability was calculated using sterile PBS-treated cells for both negative controls and experiments, all in triplicate.

Comercial human glioblastoma cells (U251 and U87), and primary glioblastoma cell line (HCB 151) derived from surgical biopsies obtained in the Neurosurgery Department of Barretos Cancer Hospital (Martinho et al., 2013 (link); Teixeira et al., 2022 (link)) (Sao Paulo, Brazil), acquired by the local ethics committee approval and the patient’s consent agreement, were kindly donated from Dr. Rui Reis, Barretos Cancer Hospital. Cell authentication was performed through short tandem repeat (STR) DNA typing, according to the International Reference Standard for Authentication of Human Cell Lines. Human Dermal Fibroblast—neonatal (HDFn) cell line was obtained from Sigma Aldrich (São Paulo, Brazil) and used as a non-malignant cell line. All cell lines were grown in Dulbecco’s Modified Eagle’s medium (DMEM; Vitrocell^® Embriolife) supplemented with 10% fetal bovine serum (FBS), gentamicin sulfate (0.05 mg/mL), and amphotericin B (25 μg/mL), L-glutamine (0.584 mg/mL) at 37°C in a humidified incubator, with an atmosphere of 95% air and 5% CO₂.