Glutamax 1

K562 stably expressing HLA-A*0201 were obtained from C Britten [23 (link)] and were maintained in IMDM (Lonza) and HEK293T cells in DMEM (Lonza). Cell culture medium was supplemented with 8% fetal calf serum (Sigma) containing 100 U/ml each of penicillin and streptomycin (Life Technologies) and 2 mM Glutamax I (Lonza) and kept in humidified incubators at 37°C containing 5% CO₂.

Seven days after serum treatment all four ankle joints were excised, the skin was removed, and the ankles were washed with sterile 70% ethanol and then with HBSS. Joints were cut into little pieces in HBSS containing 20 mM HEPES and 80 mg Liberase enzyme cocktail (Roche) and incubated at 37°C for one hour with 1400 RPM shaking. After filtration with a 70 µm pore size filter (Greiner), samples were centrifuged, the pellets were washed twice with HEPES containing HBSS, and finally suspended in 10 ml Dulbecco’s modified Eagle medium with GlutaMAX I (Lonza) supplemented with 10% (w/v) FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cells were grown in a 5% humidified CO₂ incubator at 37°C for one week on tissue culture Petri dishes (Orange Scientific). Cell lysates were Western blotted as described above and membranes were decorated with anti-ARHGAP25 and anti-GAPDH primary antibodies, and then with HRPO (horse-radish peroxidase) conjugated, rabbit IgG-specific secondary antibody (Table 1).

B16-F10 and B16-OVA murine melanoma cells were cultured at 37 °C under 5% CO2 in DMEM high glucose with GlutaMax-1 (Lonza) supplemented with 10% (v/v) foetal calf serum (Lonza), 1% penicillin, streptomycin, amphotericin B (Gibco), 4 mmol/l HEPES (Gibco) and 1 mmol/l sodium pyruvate (Gibco). B16F10 cells (ATCC CRL-6475) were obtained from American Type Culture Collection. B16-OVA cells were provided by J.D. Rosenblatt. All cells were routinely tested for Mycoplasma contamination using Mycoalert Mycoplasma Detection Kit (Lonza) and were found to be negative. To induce tumour formation, 2 × 10⁵ B16-F10 or B16-OVA cancer cells were injected subcutaneously into mice. After one week, tumour size were measured daily with an electronic calliper. According to our institutional ethical board, animals were killed by cervical dislocation, after have been anesthetised, when the maximum tumour size (2000 mm³) was achieved or when a necrosis superior to 2 mm was observed. Alternatively, 2 × 10⁵ B16-F10 or B16-OVA cells were injected intravenously into mice. Lung tumour foci were counted after 13 days by researchers ‘blinded’ to sample identity. All tumour growth experiments were approved by the Burgundy University Animal Experiments Ethics Committee (approved protocol #2212).