Fig. 3. Cancer cells enhance PD-1 protein stability in NK and T cells. (A) NK-92 and Jurkat cells were treated or not treated with CM (50%) for the indicated periods. Protein and mRNA expression levels of PD-1 were determined using immunoblotting (upper panel) and quantitative real-time PCR (bottom panel) with the indicated antibodies and primers, respectively. Data are presented as mean±SD of three independent experiments. (B) NK-92 and Jurkat cells were pretreated with DMSO or CHX (100 μg/mL) for 1 hr and then treated with CM (50%) for 6 hrs. Immunoblotting analyses were performed using the indicated antibodies. (C) NK-92 and Jurkat cells were pretreated with CM (50%) for 12 hrs and then treated with CHX (100 μg/mL) for the indicated periods. Immunoblotting analyses were performed using the indicated antibodies (left panel). Quantification of PD-1 levels relative to tubulin levels is shown (right panel). Band intensities were quantified using the ImageJ software. Data are presented as mean±SD of three independent experiments. **P<0.01, Student’s t-test. (D) NK-92 and Jurkat cells were treated or not treated with CM (50%) for 12 hrs. Immunoprecipitation analyses were performed using an anti-PD-1 antibody, followed by immunoblotting analyses using the indicated antibodies. (E) NK-92 and Jurkat cells were treated with CM (50%) for the indicated periods. The cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-PD-1 antibody, followed by immunoblotting analyses using the indicated antibodies.
Abbreviations: PD-1, programmed death 1; NK, natural killer; CM, conditioned medium; DMSO, dimethyl sulfoxide; CHX, cycloheximide.

Ann Lab Med 2024;44:426~436 https://doi.org/10.3343/alm.2023.0443