Fig 2: IL13RA2 silencing enhances cell proliferation and migration, but inhibits cell apoptosis. (A) CCK-8 assay for cell proliferation of MHCC97H-shIL13RA2 cells and HCCLM3-shIL13RA2 cells compared with their vector control. IL13RA2 knockdown increased cell proliferation of HCCLM3, but not of MHCC97H. (B) Flow cytometry analysis of apoptosis of MHCC97H-shIL13RA2 cells and HCCLM3-shIL13RA2 cells compared with their vector control. IL13RA2 knockdown decreased the total and early apoptosis rates of MHCC97H and HCCLM3 cells. (C, D) Wound-healing assays (original magnification ×40; scale bars represent 200 µm) (C) and Transwell migration assays (original magnification ×100; scale bars represent 100 µm) (D) for cell migration in the indicated group. IL13RA2 knockdown showed obvious promotion of migration abilities in MHCC97H and HCCLM3 cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by Student’s t-test. Error bars represent SD. n = 3 independent experiments per condition. 7-AAD, 7-aminoactinomycin D.

Fig 3: The expression of IL13RA2 in HCC. (A) TCGA analysis of IL13RA2 expression in human HCC and normal hepatic tissue. IL13RA2 expressed higher in normal hepatic tissue. (B) Quantitative RT-PCR analysis of IL13RA2 mRNA expression in HCC cell lines and normal hepatocyte cell line (L02). The IL13RA2 mRNA expressed higher in MHCC97L cells, MHCC97H cells and HCCLM3 cells. (C) Western blot analysis of IL13RA2 protein expression in HCC cell lines and normal hepatocyte cell line (L02). The IL13RA2 expressed higher in MHCC97H cells and HCCLM3 cells. *P < 0.05; ***P < 0.001 by Student’s t-test. Error bars represent SD. n = 3 independent experiments per condition.

Fig 4: Immunoreactive IL-13RA2 in human and canine brain tumor specimens/cells by purified MAb’s of Peptide 1 A, Human glioblastoma (G), B, oligodendroglioma (O), astrocytoma (A), normal brain (NB), and G14 human GBM tumor lysate; meningioma (M), C, tissue lysates immunoreactivity using Western blots. Canine astrocytoma, glioblastoma and normal brain, D; oligodendroglioma, gliosarcoma (GSO) and mixed astro-oligo (AO), E; and choroid plexus papilloma (CPP) and meningioma, F tissue lysates immunoreactivity using western blots. Western blot of cell lines and parent tumor tissue obtained from dogs with spontaneous GBM, G. Immunoprecipitation of IL-13RA2 from U-251 MG cell lysate using either MAb 2G12C3, MAb 2G12E2 or a polyclonal antibody (R&D Systems #AF146); the polyclonal antibody was used for the receptor detection after immunoprecipitation, H.

Fig 5: Vitreous induced the protein expression of IL13Ra2. Western blot analysis of the vitreous-induced protein expression of IL13RA2 in ARPE-19 cells. One of three representative experiments is shown. Bar graphs showed the Western blot band intensity, and the mean ± SD of three independent experiments is shown; *** denotes 0.001, using paired t test. (A) The protein expression of IL13RA2 in ARPE-19 cells. (B) Histogram of protein expression gray value. The mean ± SD of three independent experiments is shown; *** denotes 0.001, using paired t test. Control: ARPE-19 cells treated with DMEM/F12 only. Vitreous: ARPE-19 cells treated with vitreous diluted in DMEM/F12.