Fig 1: LRIG3 and sLRIG3 inhibit migration and invasion of glioma cells. (A) Wound-healing assay of GL15, U87, and PriGBM cells overexpressing full-length LRIG3 and LRIG3 ectodomain proteins (sLRIG3). Representative images of wounded cell monolayers. Scale bar, 100 μm. (B) Quantification of healing areas of the different groups from each cell lines (Data represent the mean ± SD of triplicates from healing areas of one experiment. *p < 0.05; **p < 0.01 vs. control group; one-way ANOVA). (C) Invasion capacity as measured by transwell invasion assays. Representative images of the microscopic fields are shown. Scale bar, 50 μm. (D) Numbers of migrated cells per microscopic field were analyzed from five predetermined fields (Data represent the mean ± SD. *p < 0.05; **p < 0.01 vs. control group; one-way ANOVA).
Fig 2: LRIG3 and sLRIG3 inhibit the phosphorylation of MET and components of the downstream PI3K/Akt pathway. (A) Western blot analysis of cell lysates from GL15 and PriGBM cells overexpressing full-length LRIG3 and LRIG3 ectodomain proteins (sLRIG3) for FLAG, PDGFRα/pPDGFRα, EGFR/pEGFR, MET/pMET, and Akt/pAkt. α-Tubulin was used for loading control. (B) Analysis of quantification of the bands intensity of pPDGFRα, pEGFR, pMET, and pAkt from three repeated experiments (**p < 0.01 vs. control group; one-way ANOVA). (C) Immunoblot analysis of mTOR/pmTOR and cyclin D1 in GL15 cells. α-Tubulin was used for loading control. Analysis of quantification of the bands intensity was shown in the right panel (**p < 0.01 vs. control group; one-way ANOVA). (D) GL15 cells with different LRIG3 expression status treated with rhHGF (20 ng/ml) for 15 min. Western blot analysis was carried out for FLAG, MET/pMET, Akt/pAkt, and mTOR/pmTOR. (E) Immunoblot analysis of LRIG3, pMET, and pAkt in 16 glioma tissue samples. Normal control represents one normal frontal brain tissue obtained from decompression surgery of a patient with traumatic brain injury. (F) Correlation analysis of the band intensity of pMET and pAkt with LRIG3 in the 16 glioma specimens based on densitometry of immunoblots from (E) (p-values were determined by Pearson correlation analysis; r refers to the correlation coefficient).
Fig 3: LRIG3 expression levels are correlated with the prognosis of glioma patients. (A) Representative IHC staining of LRIG3 protein expression in human glioma tissue sections of different WHO grades. Scale bars, 100 μm. (B) Immunostaining scores of LRIG3 in the 65 paraffin-embedded sections of different grade glioma samples. Data represent the mean ± SEM; *p < 0.05; **p < 0.01; one-way ANOVA. (C) Kaplan–Meier survival curves showing overall survival of the 65 patients with grades I, II, and IV gliomas. (D) Kaplan–Meier survival curves of grade III glioma patients with different LRIG3 expression levels. (E) Kaplan–Meier survival curves of grade IV glioma patients with different LRIG3 expression levels. (F) Kaplan–Meier survival curves of HGG patients with different LRIG3 expression levels. Log-rank test was used for survival analysis.
Fig 4: LRIG3 and sLRIG3 decrease tumorigenesis of glioma cells in vivo. (A) Images of nude mice bearing GL15 xenografts overexpressing LRIG3 and LRIG3 ectodomain (sLRIG3) and the control GL15 xenografts on day 60 after implantation. The harvested tumors were presented in the lower panel. (B) Tumor growth curves were plotted according to the monitored tumor size every 10 days for 60 days. Data represent the mean ± SD; n = 5; *p < 0.05, **p < 0.01; one-way ANOVA. (C) IHC staining of FLAG, pMET, pAkt, and Ki67 in transplanted tumors. Representative photographs for each antibody and each group are shown. Scale bars, 50 μm.
Fig 5: LRIG3 and sLRIG3 decrease proliferation and anchorage-independent growth of glioma cells. (A) Analysis of proliferation rates with CCK-8 in GL15, U87, and PriGBM cells overexpressing LRIG3 and LRIG3 ectodomain proteins compared with that in the control cells. (B) Anchorage-independent proliferation with colony formation assays of GL15, U87, and PriGBM cells. Representative images of soft agar colonies in each group are presented. Scale bars, 500 μm. (C) Quantitative analysis of colony numbers per well of each group (Data represent the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01 vs. control group; one-way ANOVA).
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