FUT8 Knockdown and Phenotypic Selection

The cell model employed in this work was developed previously by our research group [41 (link)]. Briefly, 96-well plates containing 1.6 × 10⁴ cells were seeded and incubated overnight at 37 °C in an atmosphere of 5% CO₂ in a humidified incubator. Next, 110 μL/well of hexadimethrine bromide (Sigma-Aldrich) at a final concentration of 8 μg/mL and 15 μL/well of two lentiviral particles from Sigma-Aldrich containing pLKO.1 plasmids targeting human FUT8 (MISSION lentiviral Transduction Particles pLKO.1-puro-CMV-TurboGFP, TRCN0000035952, and TRCN00000229959) or a non-targeting control (MISSION^® pLKO.1-puro-CMV-TurboGFP, SHC003), also from Sigma-Aldrich, were added. The cells were incubated overnight to allow transfection. Next, the culture medium was replaced with fresh DMEM containing 5 μg/mL of puromycin (Sigma-Aldrich). This medium was replaced every 72 h until resistant clones grew up. After successful lentiviral transfection, phenotypic selection of FUT8-knockdown clones was achieved via prolonged exposure to Lens culinaris agglutinin lectin (LCA) by supplementing complete medium with 500 μg/mL from Vector Laboratories (Peterborough, UK). Clones were forced to remain in this LCA-containing medium for 7 days, and after this passage, cells were seeded in complete medium without LCA.