Fig 1: Inhibition of IMP3 expression affects tumourigenic properties. (A) IMP3 siRNA or si-control were transfected into U87 and U251 cell lines to inhibit IMP3 expression, and then IMP3 expression was confirmed by western blot. (B) Cell proliferation was determined in U87 and U251 cells by MTT assay. (C) Colony formation assay was used to determine the proliferation capacity of U87 and U251 cells. Upper: images of colony formation assay (200× magnification); Lower: quantification data. (D and E) We used 10 nude mice (5 for each group) to investigate the role of si-IMP3 in the growth of U87 cells. Tumor volumes were measured with calipers every 3 days. Following post-inoculation 21 days, we measured the mean volume and weight of xenograft tumors. The data were represented by mean ± SD.*P<0.01, vs their respective controls.
Fig 2: IMP3 expression promotes migration and invasion in vitro. Mitomycin C pretreated (10 µg/ml for 30 min) U87 (A) and U251 cells (B) were incubated with serum scarce media for 24 h, scratch was made and multiple images were collected in different time interval. Cell invasion assay was determined in U87 and U251 cells (C and D) by Transwell assay. All cells were transfected with IMP3 plasmids, IMP3 siRNAs, or their controls. The data were represented by mean ± SD. *P<0.01, vs their respective control.
Fig 3: Ectopic expression of IMP3 promotes tumourigenic properties. (A) IMP3 plasmids or vector control were transfected into U87 and U251 cell lines to overexpress IMP3, and then IMP3 expression was confirmed by western blot. (B) Cell proliferation was determined in U87 and U251 cells by MTT assay. (C) Colony formation assay was used to determine the proliferation capacity of U87 and U251 cells. (D and E) We used 10 nude mice (5 for each group) to investigate the role of IMP3 over-expression in the growth of U87 cells. Following post-inoculation 21 days, we measured the mean volume and weight of xenograft tumors. The data were represented by mean ± SD.*P<0.01, vs their respective controls.
Fig 4: IMP3 expression in glioma tissues and cells. (A) Representative IHC images showed IMP3 expression was decreased in normal brain tissues compared with primary glioma samples; original magnification: 400×. Western blot was performed to measure the expression of IMP3 protein in representative glioma tissues, and normal brain tissues (B), normal NHA cell, LN-18, U87, SHG-44, U251, and U373 cells (C). The expression levels of IMP3 proteins were normalized to GAPDH in each sample. Data are presented as mean ± SD of at least three independent experiments or 3 cases of representative samples. *P<0.01 vs N.T. or NHA;**P<0.01 vs low grade; “N.T.”=normal tissues; “C.T.”=cancer tissues.
Fig 5: IMP3 affects the survival of nude mice and EMT biomarkers in U87 cells. (A) The survival rates of nude mice containing U87 cells with IMP3 over-expression or IMP3 silencing were determined as follows: 100%×(number of survivors)/(number of challenged mice). (B) Western blot analysis was used to assess the levels of E-cadherin, N-cadherin, vimentin, snail, slug and MMP9 in U87 cells with over-expression of IMP3. (C) Western blot analysis was used to assess the levels of E-cadherin, N-cadherin, vimentin, snail, slug and MMP9 in U87 cells with IMP3 siRNAs. Data are presented as Mean ± SD of at least three independent experiments or 3 cases of representative samples. *P<0.01 vs their respective controls.
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