G7513

rHypoE11 (rat) neuronal cell lines purchased from American Type Culture Collection (ATCC) was conditionally immortalized by transfer of a temperature-sensitive simian virus 40 large tumors (SV40 T) antigen to primary hypothalamic neuronal cell cultures obtained from fetal mice on embryonic days E15, E17, and E18, and from E18 fetal rats [28 (link)]. Cells were cultivated in 1× Dulbecco’s modified Eagle’s medium (DMEM, D5796, Sigma-Aldrich, Saint-Quentin Fallavier, France) with 10% fetal bovine serum (CVFSVF0001, lot: S52751-2262, Eurobio, Courtaboeuf, France), 1% penicillin/streptomycin (P4333, Sigma-Aldrich), and maintained at 37 °C with 5% CO₂. For homogeneity, the same serum was used throughout all experiments. The cells grew to form a monolayer culture attached to the culture plate and were split when they reached 70–90% confluence, with a plate ratio of 1:5. Because standard DMEM does not contain vitamin B12, methyl donor deficiency was induced by using a poor medium (DMEM D2429, Sigma-Aldrich) lacking vitamin B9 (folic acid), with the addition of 2 mM glutamine (G7513, Sigma-Aldrich), 3.7% sodium bicarbonate (S8761, Sigma-Aldrich), 0.35% glucose (G8769, Sigma-Aldrich), 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were kept in B9-free conditions for 24 or 48 h before subsequent analyses.

Promyelocytic human leukaemia (HL-60) cells were grown in RPMI-1640 medium (R5886, Sigma-Aldrich, St. Louis, MO, USA) supplemented with heat-inactivated foetal bovine serum (Linus, S01805, Madrid, Spain), L-glutamine 200 mM (G7513, Sigma-Aldrich, St. Louis, MO, USA) and 1× antibiotic–antimycotic solution (A5955, Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂. Cultures were plated at 2.5 × 10⁴ cells/mL density in 10 mL culture bottles and passed every 2 days.

Proximal murine tubular epithelial (MCT) cells were cultured in RPMI 1640 (R0883, Sigma, MO, USA) supplemented with 10% decomplemented fetal bovine serum (FBS, F7524, Sigma, MO, USA), glutamine (2 mmol/L, G7513, Sigma, MO, USA), and penicillin/streptomycin (100 U/ml; P0781, Sigma, MO, USA) in 5% CO₂ at 37°C. MCT cells were stimulated with Hb (0–500 µg/ml, corresponding to 0–30 µM of heme molar equivalents) (H0267, Sigma) and hemin/heme (0–30 µM) (H9039, Sigma). Some cells were pre-treated with sulforaphane (SFN, 2 μM, Cayman Chemical) or tert-butyl hydroquinone (tBHQ, 1 µM) (112941, Sigma) for 16 h before Hb/heme stimulation.

F11 cells (08062601; ECCAC, Salisbury, England, UK) were grown in Dulbecco’s Modified Eagle’s Medium without sodium pyruvate (DMEM; D5671; Sigma-Aldrich) supplemented with 10% (v/v) non-dialyzed fetal calf serum (F9665; Sigma-Aldrich), 100 IU/ml penicillin and 100 μg/ml streptomycin (P0781; Sigma-Aldrich), and 2 mM glutamine (G7513; Sigma-Aldrich) in a humidified atmosphere containing 5% carbon dioxide, at 37 °C. F11 cells were routinely checked for mycoplasma contamination and tested negative.

Whole blood was collected in EDTA-treated tubes and mononuclear cells were isolated by Ficoll-Hypapue density gradient centrifugation. For infection with Epstein-Barr virus (EBV), mononuclear cells were exposed to supernatants of the B95.8 cell line according to standard procedures20 . Cells were cultured in Iscove’s Modified Dulbecco’s Medium (13390, Sigma-Aldrich) supplemented with 20% fetal bovine serum (CH30160,03, Hyclone) and 1%L-glutamine (G7513, Sigma-Aldrich).

The papillary thyroid cancer cell line BCPAP and the anaplastic thyroid cancer cell line CAL-62 were obtained from ATCC and cultured in RPMI 1640 medium (R0883; Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, F9665), 2 mM glutamine (G7513; Sigma-Aldrich), 100 units/mL penicillin and 0.1 mg/mL streptomycin (P0781; Sigma-Aldrich). Cells were maintained at 37 °C with 5% CO₂ in a humidified environment. Hypoxic conditions were achieved by incubating cells in a hypoxia modular incubator chamber (Billups-Rothenberg, Inc., San Diego, CA, USA) where a 1% oxygen mix was flushed in for 4 minutes according to the manufacturer’s instructions.
BCPAP cells (10⁵ cells/mL) were treated for up to 72 h at 37 °C with CAL-62 EVs. At the end of incubation time, cells were washed three times with PBS and counted using trypan blue exclusion assay (Abcam, Cambridge, UK) in an automated cell counter (TC20TM automated cell counter, Bio-Rad, Stockholm, Sweden). The same protocol was used also for mRNA, miRNAs, and Western blot analysis.