Streptomycin sulphate

All cell lines used in the study were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Human breast adenocarcinoma MDA-MB-231, prostate carcinoma DU-145, cervix carcinoma HeLa, osteosarcoma U2-OS, glioblastoma U373, U-87 MG and T98-G, and telomerase-immortalised retinal epithelium RPE-1-hTERT cell lines were cultured in high-glucose (4.5 g/l) DMEM. Media were supplemented with 10% foetal bovine serum (Gibco/Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin, and 100 μg/ml streptomycin sulphate (Gibco/Thermo Fisher Scientific, Waltham, MA). Cells were kept at 37 °C under 5% CO₂ atmosphere and 95% humidity.
The mouse TC-1 cell line was obtained in vitro by co-transfection of murine lung C57BL/6 cells with HPV16 E6/E7 and activated human H-Ras (G12V) oncogenes. TC-1 cells were cultured in RPMI-1640 medium supplemented with 10% foetal calf serum, l-glutamine, and antibiotics³⁵ (link). Prostate carcinoma cell line TRAMP-C2³⁶ (link) was maintained in DMEM medium (Sigma-Aldrich, St. Louis, MO) supplemented with 5% FCS, Nu-Serum IV (5%; BD Biosciences, Bedford, MA), 5 µg/ml human insulin (Sigma-Aldrich, St. Louis, MO), dehydroisoandrosterone (DHEA, 10 nM; Sigma-Aldrich, St. Louis, MO), and antibiotics³⁷ (link).

Patient and control fibroblasts were grown in 37°C in humidified 95% air/5% CO₂ in Dulbecco’s modified essential medium (DMEM) containing: glucose (4.5 g/l), l-glutamine (2 mm), fetal bovine serum (FBS) (10%, v/v), penicillin (100 U/ml), streptomycin sulphate (100 μg/ml) (Gibco, Life Technologies) and Normocin (Invitrogen), sodium pyruvate (1 mm) and uridine (50 μg/ml). Control fibroblasts were obtained from healthy paediatric individuals that were matched in age with the subjects. For transfections, cells were seeded at 80% confluency in the above media formulation without antibiotics and transfected with 263 ng/cm² of wild-type CRLS1 or LYRM4 in a pCMV vector (Twist Bioscience) using FuGene HD (Promega) + Lipofectamine LTX (Invitrogen) (1:1) in OptiMEM (Invitrogen) and incubated for 48 h.

Blood was collected at 12-, 14-, and 16-weeks post-inoculation. Density gradient centrifugation was employed to separate peripheral blood mononuclear cells (PBMCs). Cells were seeded in a 24-well plate at a density of 2x106 cells/ml in a complete medium consisting of RPMI 1640 (Gibco, Grand Island, NY), 10% fetal calf serum (Atlanta Biologics, Atlanta, GA), 100 U/ml of penicillin G sodium (Gibco), 100 μg/ml of streptomycin sulphate (Gibco), 0.25 μg/ml of amphotericin B (Gibco), and 2 mM L-glutamine (Gibco).The following in vitro treatments were conducted in triplicate wells for each animal: PBS (negative control), pokeweed mitogen (10 μg/ml; Sigma), and avium PPD (10 μg/ml; Thermo Scientific). For gene expression analysis, plates were removed at 24 h and cells were harvested for RNA extraction. Cells were also harvested for flow cytometric analysis from a replicate set of plates.

3T3-L1 (RRID_0123) cells were maintained in high glucose DMEM (SIGMA, D5796) supplemented with 10% foetal bovine serum (FBS, Gibco, 10270), MEM non-essential amino acids, 1 mM Sodium Pyruvate (Gibco, 11360-039), 100 µg/µl penicillin and 100 µg/µl streptomycin sulphate (Gibco, 15140-122). Cells were plated at ~10% confluency and always split by 60% confluency to maintain differentiation capacity. Adipogenesis was induced two days post-confluency by replacing growth medium with induction medium containing 0.5 μM insulin (SIGMA, I1882), 1 µM dexamethasone (SIGMA, 04902), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, SIGMA, I7018), and 1 μM rosiglitazone (Abcam, Ab 120762). Induction medium was replaced after two days with maintenance medium containing growth medium supplemented with insulin and rosiglitazone only. Stable 3T3-L1 shRNA lines for knocking down of Tmem120a and Tmem120b genes were described in^{21 (link)}.

The kidney cancer cell lines (ACHN, 786-O, OS-RC-2, and 769-P) as well as the HK-2 cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States). These cell populations were cultivated in RPMI-1640 medium (Gibco, Carlsbad, USA) with the addition of 10% fetal bovine serum (Gibco, Carlsbad, USA), 100 mg/mL streptomycin sulphate, and 100 μL/mL penicillin (Gibco, Carlsbad, USA).

PBMCs for restimulation assays described below were isolated from heparinized cattle blood by Ficoll^® Paque Plus 1.077 (GE Healthcare Life Sciences, US) gradient density centrifugation. For ex vivo IFN-γ ELISPOT assay, cells were washed and resuspended in serum-free medium (AIM-V^®, Invitrogen). For intracellular cell staining, cells were washed twice in RPMI 1640 media (Life Technologies, UK) supplemented with 2% heat-inactivated fetal calf serum (Sigma Aldrich). After washing, cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin (Gibco, UK), and 100 μg/ml streptomycin sulphate (Gibco, UK).

MC3T3-E1 was used to investigate the in vitro cytocompatibilities of the scaffolds. The cells were cultured in α-MEM (Hyclone) supplemented with 10% fetal bovine serum (FBS; GibcoBRL, Grand Island, NY, USA) and 1% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin sulphate; GibcoBRL) at 37 °C in a humidified atmosphere with 5% CO₂, with the culture medium changed every three days.
A cell counting kit-8 (CCK-8) assay was used to analyse cell adhesion on the specimens after 6, 12 and 24 hours. The MC3T3-E1 cells were seeded at a density of 6 × 10⁴/cm² in a 48-well plate containing the specimens, with wells containing α-MEM as a negative control. The specimens and cells were co-incubated at 37 °C in a humidified atmosphere of 5% CO₂. At each time point, a volume of 40 μL of CCK-8 solution (Dojindo Molecular Technologies Inc., Kumamoto, Japan) was added to each well and incubated for 3 hours at 37 °C. Then, the OD values were read at 450 nm and 620 nm using a microplate reader (Synergy HT, Bio-tek). The mean OD obtained from the negative control was subtracted from the ODs of the test groups. The cell proliferation was also investigated using the CCK-8 assay after 1, 3, and 7 days. The seeding density of the cells was 2 × 10⁴/cm². The OD values at days 3 and 7 were normalized to those at day 1.