Fig 2: Subcellular localization of ezrin, radixin, and programmed cell death ligand-1 (PD-L1) in JEG-3 cells. Confocal laser scanning microscopy analysis for intracellular distribution of ezrin, radixin, and PD-L1 in JEG-3. In a three-dimensional reconstruction of optically sectioned JEG-3 cells (a,b), ezrin and radixin (red) were localized at the plasma membrane and preferentially colocalized with actin (green). (c,d) PD-L1 (green) was localized at the plasma membrane and preferentially colocalized with actin (red) on the plasma membrane, but not with nuclei (red). Scale bars: 20 µm. All data are representative of at least three independent experiments.

Fig 3: Schematic representation illustrating the involvement of each ERM in the regulation of PD-L1 expression in LS180 cells. Ezrin and radixin contribute to the plasma membrane localization of PD-L1 by crosslinking with the actin cytoskeleton, possibly serving as scaffold proteins without any effect on the transcriptional level. In contrast, moesin negatively regulates PD-L1 expression at the mRNA level and interacts with the PD-L1 protein. Therefore, among the ERM proteins, ezrin and radixin may function as essential scaffold proteins responsible for the plasma membrane localization of PD-L1 in LS180 cells.

Fig 4: Immunoprecipitation analysis to detect the protein–protein interactions between programmed cell death ligand-1 (PD-L1) and ezrin, radixin, and moesin (ERM) in LS180 cells. The whole-cell lysates of LS180 cells were immunoprecipitated with an anti-PD-L1 antibody or its isotype control antibody. Images of representative Western blots probed for the presence of ezrin, radixin, moesin, β-actin, and PD-L1 in the whole-cell lysates (input) and the immunoprecipitates (IP) pulled down with a control IgG or an anti-PD-L1 antibody. Molecular weights are indicated in kDa. The images are representative of three independent immunoprecipitation experiments using at least three independent replicates of protein extracts.

Fig 5: Immunoprecipitation analysis to detect the protein–protein interactions between programmed cell death ligand-1 (PD-L1) and ezrin/radixin/moesin (ERM) in HeLa cells. The whole-cell lysates of HeLa cells were immunoprecipitated with antibodies against (a) PD-L1, (b) ezrin, (c) radixin, (d) moesin, or control antibody. Western blot images of ezrin, radixin, and moesin as well as PD-L1, in addition to ß-actin, in the whole-cell lysates (input) and those co-immunoprecipitated (IP) with a control IgG or each antibody, are shown. Molecular weights are indicated in kDa.