Fig 2: Detection of Th1-type cytokines in serum during early parasite infectionConcentrations of IL-2 (A), TNF-a (B) and IFN-? (C) in serum were determined by FACS with a multi-analyte flow assay kit. Serum was acquired from three different mouse groups (RBCs, P.y-WT and P.y-GPC3 treated mice, n = 5/group) at four different time points. The values shown represent the mean and standard errors of the means. Statistical differences between groups are indicated by the P values (*P = 0.05, **P = 0.01, ***P = 0.001). The above data is a representation of four repeated experiments.

Fig 3: GPC3 protein is highly expressed in Hepa1-6 cells and Hepa1-6 cell-induced HCC tissues in mice(A) Western blot analysis of GPC3 protein in Hepa1-6 cells. (B) Localization of GPC3 in Hepa1-6 cells using confocal microscopy. (C) Immunohistochemical analysis of GPC3 in the HCC tissues. The above data is a representation of four repeated experiments. Scatter plot show the mean percentage ± SD of GPC3 positive Hepa1-6 cells. Statistical differences between groups are indicated by the P values (*P = 0.05, **P = 0.01, ***P = 0.001) and each symbol stands for the percentage of intended cells in an individual microscopic field. SM: subcutaneous model, OM: orthotopical model. Bars: 50 µm.

Fig 4: Identification of cytotoxic T cell responses in mice with P.y-GPC3 immunization(A) Schedule of Plasmodium-based vaccination in three groups of mice (RBCs, P.y-WT and P.y-GPC3 treated mice, n = 4/group). (B) Identification of GPC3-specific CTLs by IFN-? ELISPOT assays using purified CD8+ T, and BM-DCs pulsed with GPC3 protein as target cells (***P = 0.001). Representative data are shown. The data shown are the mean SD of IFN-? pots per 4 × 105 CD8+ T cells for four mice per group. ELISPOT assays were performed in triplicate and these data are representative of four independent experiments.

Fig 5: GPC3 served as a direct target of miR-4510. A In 10 cases of GPC3-GC tissue and 10 cases of GPC3-negative GC tissue, QPCR was used to detect the change in miR-4510. B KATO III and AGS cell lines were transfected with Scramble and miR-4510 mimic for 48 h, and the expression of GPC3 was detected at the protein level. C SNU-5 cells were transfected with Scramble, miR-4510 inhibitor and miR-4510 inhibitor + GPC3 OE for 48 h, and then injected in the tail vein of nude mice. D Green fluorescent protein (GFP) + cells were sorted from the lung of Scramble, miR-4510 inhibitor and miR-4510 inhibitor + GPC3 OE mice, and the expression of miR-4510 and GPC3 was detected. E SNU-5 cells were transfected with CON, normal GPC3-3'-UTR, mutant GPC3-3'-UTR, and the change in luciferase intensity was detected