Fig. 2: Screening of CADA analogs by RELITE assay to identify PD-L1 expression inhibitors.

a Schematic view of the IRES-vector bearing the FLuc equipped with the SP of PD-L1 protein, followed by the cytosolic Renilla luciferase (RLuc). b GB138 cells were grown in 60 mm plates and transiently transfected with the IRES-vector shown in (a). After 24 h, cells were split in 96-well plates and treated with a library of CADA analogs at 20 μM for 6 h. FLuc/RLuc ratio values are shown in the aerogram by using a color scale from white up to magenta (high). c Native (SPn) and mutated (SPm) amino acidic sequences of the PD-L1 SP. d GB138 cells stably expressing C-terminally HA-tagged PD-L1 protein equipped with SPn or SPm were treated with selected CADA analogs (BL458, BL445 or BL390) for 24 h at 20 μM. Total cell extracts were separated by SDS-PAGE, and PD-L1-HA was revealed using an anti-HA antibody. Calnexin (ER marker) protein level was used as a loading control. e, f GB138 cells stably expressing _SPnPD-L1-HA were treated with selected CADA analogs (BL458, BL445 and BL390) at scaling concentrations from 0 to 20 μM for 24 h. PD-L1-HA protein levels are shown in the graph, and the IC₅₀ value is indicated with a dashed line. g GB138 cells were treated with 20 μM CADA analogs (BL458 and BL466) for 24 h, and the endogenous PD-L1 protein expression level was analyzed by SDS-PAGE. h Effect of BL458 titration on the endogenous PD-L1 protein level. The IC₅₀ value is shown with a dashed line. i GB138 cells were transiently transfected with SPn- or SPm-PD-L1-HA vectors and treated with BL458 at 20 μM for 24 hours. PD-L1-HA protein levels were analyzed by SDS-PAGE. Calnexin (ER marker) protein level was used as a loading control. Data are presented as mean values +/− SD. N = 3 independent experiments. j Vulcano plots showing significantly down-(blue dots) and up-(red dots) regulated proteins in GB138 cells after treatment with BL458 molecule at 0.2, 2, and 20 µM for 24 h.