Src inhibitor 1

NT2D1 embryonal carcinoma cell were purchased from the ATCC. This cell line was cultured in DMEM (Sigma Aldrich, cat. D6546, St Louis, MO, USA) supplemented with 10% Foetal Bovine Serum (FBS Gibco, cat. 10270, Gland Island, NY, USA), L-Glutamine (Sigma Aldrich, cat. G7513) and penicillin/streptomycin (Sigma-Aldrich, cat. P0781). The cells were used from passage 15 to 35. Mycoplasma testing was routinely done with the N-GARDE Mycoplasma PCR Reagent set (Euro-Clone, cat. EMK090020, Milano, Italy).
The cells were treated with 40 ng/mL of HGF (Human Recombinant HGF, R&D Systems, cat. 294-HG, Minneapolis, MN, USA), 50 µM of a c-MET specific inhibitor (PF04217903; Sigma-Aldrich, cat. SML0263), or 5 µM of a Src inhibitor (Src Inhibitor-1; Sigma-Aldrich, cat. S2075).
We tested different concentrations (1 µM to 10 µM) of Src Inhibitor-1 on cell viability. 5 µM, was the highest concentration without toxic effect evaluated by trypan blue exclusion test, while, PF04217903 was used as already described in a recent work [21 (link)].

Human Umbilical Vein Endothelial Cells (Lonza) were amplified in a 37°C incubator with 5% CO2 in endothelial growth media-2 (EGM-2, Lonza) with 5% FBS and 5 ng/ml rhFGF (Promega) and without heparin. Cells between passages 2 and 8 were used for all experiments. When indicated, cells were treated with 5 μM GW4869 (Sigma-Aldrich D1692), 5 μM Src Inhibitor 1 (Sigma-Aldrich S2075), 10 μM ketoconazole (Sanbio 15212-100), or an equivalent volume of DMSO for 48 h, with fresh drug or DMSO added after 24 h. When indicated, cells were treated with 100 ng/ml recombinant human VEGF165 (Peprotech 100-20) or an equal volume of water for 48 h. MDA-MB-231 cells were maintained in a 37°C incubator with 5% CO2 in DMEM with 5% FBS.

BMDCs were incubated overnight with 1 μM ova or ova ICs. The following day, BMDCs were harvested, washed and resuspended in PBS at 1 × 10⁷/ml and labelled for 20 minutes at 37 °C using CellTrace Far Red (1 μM, CTFR, Invitrogen), followed by quenching in culture media for 20 minutes at 37 °C. CTFR labelled BMDCs and CTV labelled WT OT-II CD4⁺ T cells were added to 1.5 ml tubes at 1:2 (1 × 10⁵ BMDCs: 2 × 10⁵ T cells, in a total volume of 50 μl), centrifuged for 2 minutes at 25 g and incubated at 37 °C for 0–120 minutes. Cells were fixed using 3% PFA in PBS, transferred to FACS tubes and acquired by flow cytometry using a medium flow rate. Conjugates were identified as CTV⁺ CTFR⁺ events. To identify the role of Src and Syk family kinases in conjugate formation, WT BMDCs were pre-incubated with Src inhibitor-1 (5 µM, Sigma) or Syk inhibitor II (5 µM, Calbiochem) for 15 minutes at 37 °C, prior to the conjugate assay (described above).