Fig. 2: Cancer senescence could influence glycosylation, which is crucial for PD-L1 stabilization and membrane transport.

a Immunofluorescence staining of PD-L1 and markers of ER (HSP90B1) and Golgi (cis, GM130) in Control (Cont) or IR-induced cancer senescence (IR-CS). Nuclei were counterstained with DAPI. Scale bar: 10 µm. Representative images of n = 3 independent replicates. b Cell surface levels of PD-L1 were analyzed by flow cytometry in IR-induced senescent H460 cells. The gating strategies are provided in Supplementary Fig. 13a. c PD-1 binding assay: representative images showing the binding of green fluorescence-labeled PD-1/Fc protein on IR-induced senescent H460 cells. Scale bar: 50 µm. Representative images of n = 3 independent replicates. d A Venn diagram was created to compare the N-glycosyltransferase list obtained from our microarray data in this study with the list of N-glycosyltransferases known to interact with PD-L1⁴³. e Identification of N-glycosylation-related enzymes that are differentially expressed in IR-induced senescent H460 cancer cells. f Validation of increased mRNA expressions of N-glycosylation-related enzymes in IR-induced senescent H460 cells. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. g qRT-PCR analysis of PD-L1 mRNA levels in IR-induced senescent H460 cells, which were transfected with each indicated siRNAs. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. h Immunoblot analysis of PD-L1 levels in IR-induced senescent cancer cells, which were transfected with each indicated siRNAs. Representative immunoblots of n = 3 independent replicates. Statistical significance is represented as means ± SD. Source data are provided as a Source Data file.