L glutamine

Complete RPMI medium was prepared by supplementing RPMI 1640 medium (with phenol red or phenol red-free, as indicated; Invitrogen, Darmstadt, Germany) with heat-inactivated fetal calf serum (FCS, 10% v/v; PAA Laboratories, Pasching, Austria), L-glutamine (final concentration 2 mM; Biochrom AG, Berlin, Germany), HEPES (pH 7.2, 0.01 M; Invitrogen, Darmstadt, Germany), penicillin G (0.2 U/ml; Sigma-Aldrich, Taufkirchen, Germany), gentamicin (0.05 mg/ml; Sigma-Aldrich), and 2-mercaptoethanol (0.05 mM; Sigma-Aldrich). In addition, for generation of BMM, a conditioned medium was used containing Dulbecco's Modified Eagles Medium (DMEM; Invitrogen), heat-inactivated FCS (10% v/v; PAA Laboratories), heat-inactivated horse serum (0.5%; Invitrogen), 2-mercaptoethanol (0.05 mM; Sigma-Aldrich), nonessential amino acids (Invitrogen), HEPES (0.01 M; Invitrogen), L-glutamine (4 mM; Biochrom) and L929 supernatant (15% v/v). L. major promastigotes were cultured in a biphasic medium consisting of a solid base of rabbit-blood agar (Elocin-lab, Gladbeck, Germany) plus a liquid phase of RPMI medium without phenol red.

Human cutaneous melanoma cell lines (Mel 195, 275, 313, 346, 116, 120, 514, 142, 237, 403, 458, 345, 599, and 261) were generated from surgically removed metastatic lesions from melanoma patients, as previously described (Altomonte et al., 1993 (link)). Human hematological cancer cell lines (Daudi, HL-60, NALM-6, Raji, U-937, KG-1a, Jurkat, JY, Ri-1, K562) were purchased from American Type Culture Collection (Rockville, MD, United States).
Melanoma cells were grown in RPMI 1640 (Carlo Erba, Milan, Italy) supplemented with 10% heat-inactivated FBS (Biochrom, Berlin, Germany) and 2 mM L-glutamine (Biochrom, Berlin, Germany). Hematological tumor cell lines were grown in ISCOVE Basal Medium (Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine and 100 μg/μl penicillin/streptomycin (Biochrom, Berlin, Germany).

The E14 mESC line was maintained in the naive state on gelatin-coated plates in Knockout^TM DMEM (ThermoFisher Scientific) with 15% Hyclone competent serum (VWR), 2 mM L-glutamine, 1% penicillin/streptomycin (Biochrom), 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and 0.1 mM β-mercaptoethanol (all are from ThermoFisher Scientific) supplemented with LIF (1000 U; Merck) and 2i (1 μM PD0325901 and 3 μM CHIR99021; Miltenyi Biotech).
E14 mESCs were also adapted to serum-free medium containing a 1:1 mixture of advanced Dulbecco’s modified Eagle’s medium F12 and neurobasal medium (ThermoFisher Scientific), supplemented with 1 × N2, 1 × B27, and 40 mg ml⁻¹ BSA (ThermoFisher Scientific) plus 2 mM L-glutamine, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, and 12.5 μg ml⁻¹ insulin (Sigma-Aldrich), including LIF and 2i (Fig. 2b). Differentiation of E14 mESCs to NPCs was performed in serum-free medium on matrigel-coated (BD Biosciences) plastic dishes following the protocol described in ref. ^{59 (link)}. Briefly, E14 mESCs were cultured without the inhibitors and with 10 ng ml⁻¹ basic fibroblast growth factor (bFGF; ThermoFisher Scientific) for two days, with a combination of bFGF and 5 μM XAV (Sigma-Aldrich) for 1 day, and with XAV alone for 2 days. After six days of culture, both naive mESCs and NPCs were collected for western blotting analysis (Fig. 2a).

Human epithelial cell line HeLa were maintained in DMEM containing 4.5 g x l^-1 glucose, 4 mM L-glutamine and sodium pyruvate (Biochrom) supplemented with 10% fetal calf serum (FCS) in an atmosphere of 5% CO₂ and 90% humidity at 37°C. The murine macrophage cell line RAW264.7 (ATCC no. TIB-71) were cultured in DMEM containing 4.5 g x l⁻¹ glucose and 4 mM stable glutamine (Biochrom) supplemented with 6% FCS. For activation of RAW264.7, cells were cultured in medium with 5 ng x ml^-1 murine IFN-γ (R&D Systems) for 24 h prior to infection. The efficacy of activation was routinely confirmed by analyses of macrophages oxidative burst.