Pe isotype control

Human GC cell lines AGS, MKN-45, MKN-74, KATO III, SNU-1, and SNU-16 (5×10⁵ cells) were either directly harvested for staining or co-cultured with CF33-hNIS-Δ (MOI=3), CF33-hNIS-antiPDL1 (MOI=3), or phosphate buffered saline (PBS) (control) for 15 hours. Then, cells were harvested for surface and intracellular CD274/PD-L1 expression. For cell surface staining, cells were washed with PBS, blocked with 10% human serum in PBS, stained with PE-isotype control (Biolegend, Cat#402204, clone#27-35) or PE-anti-PD-L1 antibody (Biolegend, Cat#329706, clone#29E.2A3), washed thrice with 2% FBS PBS, and analyzed with a BD LSRFortessa Flow Cytometer (BD Biosciences, San Jose, California, USA). In the virus-treated group, cells were fixed with 4% paraformaldehyde before performing flow cytometry. For intracellular staining, cells were first washed with PBS and blocked with 10% human serum. Then the cells were fixed/permeabilized with a fixation/permeabilization solution (Catalog#554714, BD Biosciences) for 20 min, washed twice with BD Perm/Wash buffer, stained with antibodies for 30 min, and washed twice with BD Perm/Wash buffer. Stained cells were assessed with a BD LSRFortessa Flow Cytometer and analyzed using Flowjo software. Results are shown as histograms and mean fluorescence intensity (MFI).47 (link) All experiments were repeated at least three times.