Knockdown of PD-L1 and PD-L2 in CMFs

Small interfering RNA technology was used to knockdown expression of PD-Ls molecules in human primary CMF isolates as described previously (16 (link)). Breifly, negative siRNA controls are included in each experiment. Stealth siRNAs Set of three siRNA probes to the conservative domains of PD-L1, PD-L2, or negative siRNA control were purchased from Thermo Fisher Scientific. Transfection of CMFs was performed using Human Dermal Fibroblast Nucleofector kit according to the manufacturer instruction (Lonza, Allendale, NJ, USA). The efficiency of the downregulation of the PD-L1 or PD-L2 expression by specific siRNA set was controlled by real-time RT-PCR and flow cytometry.