Generating Engineered Cell Lines

To generate control, integrin β3, or ferroportin-1 overexpressing cell lines, the following plasmids were cloned by Vector Builder (Chicago, IL, USA) according to our design: mouse empty vector control (pLV[Exp]-Puro-EF1A>{Stuffer_300bp}); mouse β3 integrin OE vector (pLV[Exp]-Puro-EF1A>mItgb3[NM_016780.2]); human empty vector control (pLV[Exp]-EF1A>ORF_Stuffer-CMV>tdTomato(ns):T2A:Puro); human β3 integrin OE vector (pLV[Exp]-EF1A>hITGB3[NM_000212.2]-CMV>tdTomato(ns):T2A:Puro); and human ferroportin-1 OE vector (pLV[Exp]-EF1A>hSLC40A1[NM_014585.6]-CMV>tdTomato(ns):T2A:Puro). These plasmids were received as E. Coli glycerol stocks and plated on ampicillin-containing plates to generate single-cell colonies for expansion and plasmid isolation. The plasmid DNA was extracted from bacterial pellets using Wizard^®Plus SV Minipreps DNA Purification System kit (Promega, #A1330) as per the manufacturer’s instruction. For transfection, total plasmid DNA (2.5 μg) containing empty or integrin β3/ferroportin-1 expression vector and packaging vectors (pCMVΔR8.2, second-generation lentiviral plasmid and pCMV-VSV-G, envelop plasmid obtained form Adgene) in equimolar concentrations were added to 125 µL of Opti-MEM, followed by the addition of 5 µL of P3000 Lipofectamine Reagent. The plasmid mix was further combined with Lipofectamine 3000 Reagent pre-diluted in Opti-MEM in a 1:1 ratio and incubated at RT for 15 min to form a lipid-DNA complex that was added to the HEK293T cells with an additional 750 µL Opti-MEM. The transfected HEK293T cells were incubated for 24 h at 37 °C, 5% CO₂. The transfection medium was then replaced with 1 mL of fresh DMEM supplemented with 10% FBS (without antibiotics) for another 24 h. The lentiviral supernatant was collected and filtered through a 0.45 µm filter. After the addition of polybrene (8 µg/mL), the lentiviral supernatant was added to semi-confluent target cancer cells for 24 h. Fresh complete DMEM (1 mL) was added to the transfected HEK293T cells and harvested after another 24 h for a second round of transduction as described above. Stably transduced cells were selected either by incubation with puromycin (5 µg/mL)-containing complete medium over 7 days (for mouse cells) or by the selection of td-tomato-positive cells (for human cells) by FACS. Integrin β3 or ferroportin-1 OE was validated by Western blotting.