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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: TRIM56 promotes malignant progression of glioblastoma by stabilizing cIAP1 protein

Fig. 5

TRIM56 promotes the development of glioma by enhancing the stability of cIAP1. a-b GBM#P3-, LN229- and U118MG-TRIM56OE cells were immunoprecipitated using IgG antibody or TRIM56/ cIAP1 antibody and were immunoblotted using TRIM56 and cIAP1 antibodies. c Western blot of lysates prepared from GBM#P3-, LN229- and U118MG-TRIM56KD, -TRIM56CON and -TRIM56.OE cells treated with 25 Î¼g/mL of cycloheximide (CHX) for the indicated times to determine half-life of cIAP1 protein. Blots are incubated with the antibodies indicated d Growth curves generated from cell number counts performed on the cells over a 72-h period (at 24-h intervals). e Fluorescence images and quantification of EdU assays performed on cells (scale bar, 500 Î¼m). f Fluorescence images and quantification of staining for cleaved caspase-3. DAPI was used to stain nuclei (scale bar, 500 Î¼m). g Cell cycle analysis for the indicated cells. The percentage of cells arrested in the G0/G1 phase is analyzed in the bar graphs below. h Representative images of colony formation assays and quantification on cells shown (GBM#P3- LN229- and U118MG-shTRIM56-NC, -shTRIM56-S2 and -shTRIM56-S2 cells transfected with Myc-cIAP1) seeded at 1000 cells/well and cultured for 2 weeks. i Brightfield images of 3D tumor sphere invasion assays and quantification for GBM#P3 cells transfected with plasmid for Myc-cIAP1 or GBM#P3-shTRIM56-S2 cells. Representative images at 0 h, 24 h and 48 h are shown, as well as quantitative plots of the percentage of invaded area (scale bar, 200 Î¼m). j Representative images of co-culture invasion assays and quantification for GBM#P3 cells. Invasion was assessed at 24 h, 48 h and 72 h (scale bars = 200 Î¼m). k Representative images of Transwell assays and quantification performed on LN229- and U118MG-shTRIM56-S2 cells. Cells were fixed, stained with crystal violet and counted (scale bar, 50 μm). Comparisons between two independent samples and among multiple samples were performed using two-tailed t tests and one/two-way ANOVA, respectively. Error bars indicate at least three independent experiments, and data are shown as mean ± SEM. ∗ p < 0.05, ∗  ∗ p < 0.01, ∗  ∗  ∗ p < 0.001, and ∗  ∗  ∗  ∗ p < 0.0001

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