Luciferase reporter assay

For the cap-dependent translation assay, cells were transfected using Lipofectamine 3000 reagent with the dicistronic luciferase reporter plasmid, pcDNA3-RL-Polio IRES-FL, which directs cap-dependent translation of the RL gene and cap-independent Polio IRES-mediated translation of the FL gene.²¹ After 24 h transfection, cells were treated with AZD8055 or INK128 for 24 h. Cell lysates were prepared and assayed for RL and FL activities using a Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol. Cap-dependent RL activity was normalized against cap-independent FL activity as the internal control. The ratio of RL/FL activity was calculated for cap-dependent translational activity.²¹ To identify the presence of IRES activity in the PD-L1 5′-UTR, cells were transfected with dicistronic PD-L1 5′-UTR reporter plasmids. For PD-L1 promoter assay, cells were co-transfected with the FL reporter containing the human PD-L1 promoter along with an RL vector pHRL-TK (for normalization). Thirty-six hours post-transfection, cell lysates were prepared and analyzed similarly using the Dual-Luciferase Reporter Assay Kit (Promega). IRES- or promoter-dependent FL activity was normalized to RL activity.