Leibovitz l 15

Ethical approval for receipt and use of human fetal tissues was obtained from the South East Scotland Research Ethics Committee (LREC08/S1101/1) and NRES committee North East – Newcastle and North Tyneside 1 (08/H0906/21 + 5) and written and informed consent was provided by the pregnant women. First- and second-trimester human fetal testes (8.5–20 weeks gestation; n = 12) were obtained after medical termination of pregnancy as described previously (Mitchell et al., 2008 (link)). None of the terminations were due to fetal abnormalities. Gestation was determined by ultrasound scan and subsequent direct measurement of foot length. The sex of first trimester testes was confirmed by PCR for the male-specific gene SRY. To permit subsequent gene expression analysis, where possible a small sample of gonad tissue was removed, snap frozen and stored at −70°C until RNA extraction. The remainder was placed immediately into medium containing Leibovitz L-15 with added glutamine, 10% fetal bovine serum, 1% penicillin/streptomycin and 1% non-essential amino acids (all Sigma-Aldrich).
For studies involving animals, specific approval, including ethical approval, was given by the UK Home Office and all procedures were carried out in accordance with the Animal (Scientific procedures) Act 1986.

The human melanocyte cell line (AG21857) was obtained from the NIA Cell Culture Repository through the Coriell Institute (only used in Fig. 5B). Melanoma cell lines (WM983A and WM983B) were obtained from the Wistar repository through the Coriell Institute. These melanoma cell lines were isolated from the same patient and correspond to VGP primary melanoma (WM983A) and metastatic melanoma (WM983B). The WM983B metastatic melanoma cell line is the main cell line used throughout this study and corresponds to NTF2 low, NTF2 high dox-, and NTF2 high dox +. Anywhere that we refer to VGP primary melanoma we are using the WM983A cell line. The human metastatic prostate cancer cell line (LNCaP) was obtained from Jay Gatlin (University of Wyoming). AG21857 cells were maintained in Medium 254 (#M254500, Invitrogen) supplemented with HMGS (#S0025, Invitrogen). WM983A and WM983B cells were maintained in 4:1 MCDB153 (#M7403, Sigma-Aldrich) and Leibovitz L-15 (#L4386, Sigma-Aldrich), supplemented with 5 µg/ml insulin (#I9278, Sigma-Aldrich), 2% FBS, 1.68 mM CaCl₂, and 50 IU/ml penicillin and streptomycin. LNCaP cells were maintained in RPMI-1640 (Sigma) supplemented with 7.5% FBS and 50 IU/ml penicillin and streptomycin. Tet-free FBS was used for experiments where NTF2 expression was induced with doxycycline. All cells were grown at 37 °C and 5% CO₂.

Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. Leibovitz (L15); streptomycin; penicillin; DMSO; docetaxel; hematoxilin were purchased from Sigma-Aldrich (St. Louis, MO, USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazoliumbromide); propidium iodide; Anexina-V cj FITC; Alexa-488 were purchased from Invitrogen. Osmium tetroxide; Spurr resin were purchased from Electron Microscopy Sciences.

Cytotoxicity and AhR agonist activity were investigated using the permanent cell line RTL-W1 derived from rainbow trout (Oncorhynchus mykiss).[50 (link)] Cells were maintained in 75 cm² cell culture flasks in L-15 (Leibovitz; Sigma-Aldrich) medium supplemented with 9% fetal bovine serum (FBS; Biowest, Nuaillé, France) and 1% penicillin/streptomycin solution (Pen/Strep, with 10,000 units penicillin and 10 mg streptomycin per ml in 0.9% NaCl; Sigma-Aldrich). RTL-W1 cells were kept in darkness at 20°C. Cells were passaged once a week in a ratio of 1:2 using 1x trypsin/EDTA (Sigma-Aldrich).

The TiLV strain (VETKU-TV01) was isolated from infected red hybrid tilapia in Pathum Thani province, Thailand in 2016 [30 (link)]. TiLV was propagated in snakehead fish (Ophiocephalus striatus) E-11 cells [74 (link)]. The E-11 cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC #01110916) and maintained in L-15 Leibovitz (Sigma, Cambridge, MA, USA) medium supplemented with foetal bovine serum (Gibco, Paisley, UK) and L-glutamine at 25 °C, without CO₂, and without the addition of antibiotics. The titer of TiLV (TCID₅₀/_mL) was determined following the protocol described by Reed and Muench [75 (link)]. The cell suspension was centrifuged at 3000× g for 10 min and the supernatant was stored at −80 °C until the challenge test.