Nectin4 sirna

Cells were transfected with control siRNA (Invitrogen, Carlsbad, CA, USA; AM4611) or NECTIN4 siRNA (Invitrogen; s37689) using Lipofectamine RNAiMAX (Invitrogen; 13778075). Briefly, siRNAs were diluted with Opti-MEM^TM I Reduced Serum Medium (Thermo Fisher Scientific, Waltham, MA, USA; 31985062), mixed with Lipofectamine, and incubated for 20 min at room temperature. Then, the siRNA-Lipofectamine complexes were mixed with the cells (final siRNA concentration of 10 nM). At 24–72 h after transfection, the cells were harvested and used for further analysis. The knockdown efficiency was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting.

HAMON and ISO-HAS-B cells were transfected with either negative control siRNA (Invitrogen, Carlsbad, CA; AM4611) or NECTIN4 siRNA (Invitrogen; s37690) using Lipofectamine RNAiMAX (Thermo Fisher Scientific; 13778150) in accordance with the manufacturers’ instructions. Briefly, cells were seeded into six-well plates (2.0 × 10⁵ cells/well), 12-well plates (1.2 × 10⁵ cells/well), or 96-well plates (3,000 cells/well) and incubated for 24 h at 37 °C in 5% CO₂ before being transfected with the siRNA and incubated for a further 1–5 days at 37 °C in 5% CO₂. Cell proliferation and in vitro tube formation were then analyzed, as detailed below. NECTIN4 knockdown efficiency was assessed by qRT-PCR and western blotting, and the amount of VEGF secreted into the culture medium was determined by ELISA (see below).

To evaluate the function of NECTIN4 in SCC cells, A431 cells were seeded in 96-well plates (2000 cells per well), 12-well plates (1.2 × 10⁵ cells per well), or 6-well plates (2.0 × 10⁵ cells per well) and transfected with control siRNA (Invitrogen, Carlsbad, CA, USA; AM4611) or NECTIN4 siRNA (Invitrogen; s37689) using Lipofectamine RNAiMAX reagent (Invitrogen; 13778075) in accordance with the manufacturer’s instructions. Briefly, siRNAs were diluted in Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific; 31985062), mixed with Lipofectamine, and incubated for 20 min at room temperature. After the incubation, the siRNA–Lipofectamine complexes were mixed with the cells in culture plates (final siRNA concentration: 10 nM). The cells were harvested 1–5 days after transfection and used in further analyses. NECTIN4 knockdown efficiency was examined using quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and western blotting.