Repurposing screen identifies Amlodipine as an inducer of PD-L1 degradation and antitumor immunity

T-cell killing assay
RKO cells were treated with amlodipine for 24h and then seeded in a 96-well plate with 1×106
cells in each well. Meanwhile, human peripheral blood mononuclear cells (PBMC; #CE-003,
StemEry) were stimulated with 100ng/mL CD3 antibody (#317303, BioLegend), 100ng/mL
CD28 antibody (#302913, BioLegend) and 10 ng/mL IL2 (#589102, BioLegend) in RPMI-1640
complete medium for 24h. the pre-treated RKO cells were then co-cultured with the activated
PBMCs at 1:10 ratio at 37°C under 5%CO2 for about eight hours. The cells were co-cultured
in RPMI-1640 complete medium added fluorescence caspase-3/7 substrate (I35106,
ThermoFisher Scientific) for 60 minutes and subsequently Hoechst for 5 minutes. After washed
twice, the cells were observed by fluorescence microscopy using appropriate bandpass filters.