Fig 1: External validation of 12 immune-related genes. (A) Pearson correlation of expression and ImmuneScore using GSE62232 dataset. TRBC2 and TRBV28 were not detected. 9 genes, except TMC8, had significant correlation. Y-axis represented expression level of the genes in each sample. (B) Representative immunohistochemical staining images of 7 genes in normal liver tissue and HCC specimen. Images were taken from the Human Protein Atlas (https://www.proteinatlas.org). TMC8 was strongly positive, BIN2, GIMAP7, SPOCK2 were moderately positive, and FYB1, RCSD1, SASH3 were weakly positive in HCC tissues relative to their expression levels in normal liver tissues.
Fig 2: Measurement of TMC8, BIN2 and SPOCK2 at mRNA and protein level in our cohort. (A, B) Relative mRNA levels of TMC8 and BIN2 in 10 HCC samples were both overexpressed compared with matched normal samples by qRT-PCR. (C) Representative western blotting images showed protein expression of TMC8, BIN2 and SPOCK2 were overexpressed in HCC tissue than those in normal liver tissue. (D–F) Western blotting analysis demonstrated that mean greyscale of TMC8, BIN2 and SPOCK2 were all higher in 10 fresh-frozen HCC tissues compared with those in matched adjacent normal liver tissues. (G) Representative immunohistochemical staining images of TMC8, BIN2 and SPOCK2, which were all mainly expressed in the cytoplasm. Original magnification: x200. (H–J) Mean protein expression of TMC8, BIN2 and SPOCK2 in 35 HCC were all significantly higher compared with those in adjacent non-tumour tissue by immunohistochemistry. MAD: mean areal density.
Fig 3: SPOCK2 and SPRED1 are decreased in LUAD cells and inhibit the proliferation of LUAD cells. A The expression of SPOCK2 and SPRED1 between tumor tissues and normal tissues were analyzed using TCGA LUAD database. **P < 0.01 vs the normal group; t test; n = 59/515 (normal/tumor). B Survival analysis of SPOCK2 and SPRED1 in TCGA LUAD database. The best high and low expression cut-off values were 9.68 for SPOCK2 and 7.73 for SPRED1. SPOCK2 or SPRED1 overexpression vector and the control vector were transfected into A549 and HCC827 cells respectively, and the stable transfected cell lines were screened with G418. C The expression of SPOCK2 and SPRED1 in LUAD cells were analyzed by qRT-PCR and western blot. **P < 0.01 vs the pcDNA3.1 group; one-way ANOVA; n = 3, repeated three times. D CCK-8 assay was performed to evaluate LUAD cell proliferation. *P < 0.05, **P < 0.01 vs the pcDNA3.1 group; two-way ANOVA; n = 3, repeated five times. E The effect of overexpression of SPOCK2 or SPRED1 on colony formation of LUAD cell. **P < 0.01 vs the pcDNA3.1 group; one-way ANOVA; n = 3. All data are represented as mean ± SD
Fig 4: Overexpression of SPOCK2 or SPRED1 suppresses the migration ratio and invasion activity in LUAD cells. A The migration ratio of A549 and HCC827 cells transfected with SPOCK2 or SPRED1 overexpression plasmid was assessed by wound-healing assay. **P < 0.01 vs the pcDNA3.1 group; one-way ANOVA; n = 3; magnification, 100×. B The invasion ability of LUAD cells was evaluated by the transwell assay. **P < 0.01 vs the pcDNA3.1 group; one-way ANOVA; n = 3; magnification, 200×. All data are represented as mean ± SD
Fig 5: The antineoplastic effects of SPOCK2 or SPRED1 are impaired by EZH2. A549 cells were transfected with SPOCK2 or SPRED1 overexpression vector and/or the control vector or EZH2 overexpression vector. A CCK-8 assay was performed to evaluate cell proliferation. **P < 0.01 vs the SPOCK2 + pcDNA3.1 group or SPRED1 + pcDNA3.1 group; two-way ANOVA; n = 3, repeated five times. B The migration ratio was evaluated by wound-healing assay. **P < 0.01 vs the SPOCK2 + pcDNA3.1 group or SPRED1 + pcDNA3.1 group; t test; n = 3; magnification, 100×. C The invasion ability was tested by the transwell assay. **P < 0.01 vs the SPOCK2 + pcDNA3.1 group or SPRED1 + pcDNA3.1 group; t test; n = 3; magnification, 200×. All data are represented as mean ± SD
Supplier Page from Abcam for Anti-SPOCK2 antibody