Fig 1: PD-L2 knockdown inhibits osteosarcoma cells metastasis in vivo.a Tumor growth curves and tumor weights were evaluated between shPD-L2 and shNC groups. No significant difference was observed between the two groups. b Representative images of primary tumors. c The metastatic frequency of shNC and shPD-L2 groups. d H&E staining of the lungs of shNC and shPD-L2 groups (magnification × 16 and × 100). Arrows indicate metastases. e, f Primary tumors were collected and evaluated by IHC (magnification × 400) and western blotting. g A diagrammatic sketch of PD-L2 in osteosarcoma metastasis. Data are presented as the mean ± SD (n = 4)
Fig 2: Effect of PD-L2 expression on the proliferation of osteosarcoma cells.a PD-L2 expression in osteosarcoma cell lines was evaluated by western blotting and real-time PCR. b The osteosarcoma cell lines (red) exhibited differing degrees of PD-L2 expression compared with the isotype control (black) by flow cytometry. c KHOS and U2OS cells were stably transfected with shPD-L2 or shNC lentivirus, following evaluation of PD-L2 expression by western blotting and real-time PCR. d Cell proliferation of KHOS and U2OS cells in response to PD-L2 knockdown determined by cell colony formation assay. e Cell viability of KHOS and U2OS cells after PD-L2 knockdown assayed by CCK-8. All experiments were repeated three times. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 3: IHC staining for PD-L1, PD-L2, and PD-1 expression in the TMA samples. PD-L1, PD-L2, and PD-1 exhibited a membranous expression accompanied by cytoplasmic expression. Representative images for every histological type are shown (The background image was magnified at × 50, while the insert image was magnified at × 400 in GCT. The other histological types were magnified at × 400)
Fig 4: shSNF5 neutralizes the effects of matrix stiffness on tumor development. (a) Western blot shows the expression of p16 and RB at the protein level in A375 cells cultured on the matrixes with different degrees of stiffness with shNC or shSNF5 treatment. (b) Western blot shows the expression of ZEB1, SNAI1, VIM and N-Cad at the protein level in A375 cells cultured on the matrixes with different degrees of stiffness with shNC or shSNF5 treatment. (c) Western blot shows the expression of PD-L1, PD-L2, IDO1 and FAS at protein level in A375 cells cultured on the matrixes with different stiffness with shNC or shSNF5 treatment. (Values are shown as the mean ± standard deviation [SD.]. n = 3, a representative experiment is shown; ‘ns’ means P > 0.05, ‘*’means P < 0.05, ‘**’ means P < 0.01, ‘#’means P < 0.05, ‘##’ means P < 0.01).
Fig 5: Survival analyses according to the PD-L1, PD-L2, and PD-1 expression levels in the GEO and IHC cohorts. a OS according to PD-L1, PD-L2, and PD-1 in the GEO cohort. b OS according to PD-L1, PD-L2, and PD-1 in the IHC cohort. c Subgroup survival analyses according to PD-L1 and PD-L2 expression in the IHC cohort. d Subgroup survival analyses according to PD-L1 and PD-1 expression in the IHC cohort. e Subgroup survival analyses according to PD-L2 and PD-1 expression in the IHC cohort
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