4.2. Transfection of Cells with siRNAs

HeLa cells were cultured until 70–80% confluency in flasks and then seeded at a density of 1.5 × 10⁴ cells/well, in 24-well cell culture plates (Corning, Glendale, AZ, USA), for total RNA isolation and flow cytometry analysis; at 6.0 × 10⁴ cells/well, in 6-well cell culture plates (Corning), for total protein isolation; and at 3.0 × 10³ cells/well, in 96-well cell culture plates (Corning), for cell viability assay. The cultures were incubated overnight at 37 °C in a humidified atmosphere with 5% CO₂ to allow for cell attachment. Silencer Select siRNA for each target gene (Thermo Fisher Scientific, Tokyo, Japan), which selectively suppresses the target gene expression, was diluted with Opti-MEM (Thermo Fisher Scientific). Then, 2 nM/well or 5 nM/well of siRNA targeting human ezrin, moesin, or radixin, and PD-L1, respectively, were introduced into cells using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). The volume of transfection reagent used was 0.05 µL/well for total RNA isolation, 0.20 µL/well for total protein isolation, and 0.01 µL/well for cell viability assay. After addition of the siRNA and transfection reagent, cells were cultured continuously for 3 days without exchanging medium. Silencer Select nontargeting control siRNA (Thermo Fisher Scientific), which has no significant similarity to human gene sequences and has minimal effects on gene suppression, was used as the nontargeting control for each siRNA.