Fig 2: D-mannose synergizes with PD-1 antibody to inhibit TNBC growth. (A) Schematic representation of the animal experiment process. (B) Representative tumors of 4T1 cells in BALB/c mice treated with D-mannose or/and anti–PD-1 antibody. (C) Tumor growth of 4T1 cells in BALB/c mice following treatment with D-mannose or/and anti–PD-1 antibody was determined. Tumors were measured at the indicated time points. n = 8 mice per group. Statistical differences were determined by ordinary one-way ANOVA. ns, no significance, ****P < 0.0001. (D) Kaplan–Meier survival curves for mice injected with 4T1 cells and treated with D-mannose or/and anti–PD-1 antibody. The P value, comparing every two groups, was determined using log-rank test and is shown in the table Below the figure. (E) Western blot analysis of PD-L1 level in 4T1 tumor tissues as indicated. (F) Immunohistochemistry showing CD8+ T cell infiltration and granzyme B expression in the 4T1 tumor tissues as indicated (Scale bars, 100 μm). (G) Quantifications of images in (F). Data represent mean ± SD from six independent samples of each group. Statistical differences were determined by ordinary one-way ANOVA. ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: 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 4: Gene and protein expression patterns of programmed cell death ligand-1 (PD-L1) and ezrin, radixin, and moesin (ERM) in JEG-3 cells. (a–d) Representative amplification curves of (a) PD-L1, (b) ezrin, (c) radixin, and (d) moesin together with ß-actin (internal control) in JEG-3 cells as determined using real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). (e) Typical western blotting images of PD-L1 with ezrin, radixin, and moesin, as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in whole cell lysates of JEG-3 cells. HeLa cells were used as positive controls as they abundantly express moesin protein. Molecular weights are indicated in kDa.

Fig 5: Schematic diagram of a proposed model illustrating the different functions of ezrin and radixin in the regulatory mechanism of programmed cell death ligand-1 (PD-L1) expression in JEG-3 cells. Ezrin contributes to the cell surface expression of PD-L1, possibly via protein–protein interactions, without influencing its mRNA. Despite the presence of protein–protein interactions between PD-L1 and radixin, radixin may have no impact on either mRNA or the cell surface expression levels of PD-L1. Therefore, among ERM proteins, ezrin may play an essential role as a scaffold protein, contributing to the cell surface plasma membrane localization of PD-L1 in JEG-3 cells. Arrow indicates the reduction of PD-L1 expression.