PD-L1 Knockdown via siRNA Transfection

To decrease PD‐L1 expression (KD), cells were transiently transfected with small interfering RNA (siRNA) directed against the human PD‐L1 gene CD274. Dharmafect‐1 (Thermo Scientific, Waltham, MA, USA) was used as transfection reagent according to the manufacturer's instructions. Transfection was performed in 6‐well plates (Corning, NY, USA). A cell suspension of 2 × 10⁵ cells was added to prepared transfection complexes. Transfection with 25 nm siRNA was carried out in DMEM growth medium without antibiotics for 72 h. The siGENOME Human CD274 siRNA SMARTpool (M‐015836‐01‐0005, Horizon Discovery, Cambridge, UK) consists of four siRNA target sequences. This method has already been described in [21 (link)]. The ON‐TARGETplus Non‐Targeting Control Pool (D‐001810‐10‐20, Horizon Discovery) was used as control. The siRNA sequences used for PD‐L1 KD experiments are listed in Table 1.