Fig. 2

KPNB1 knockdown inhibited glioblastoma growth, which could be rescued by overexpression of KPNB1. A. Western blot analysis showing KPNB1 expression in U87MG and U251MG cells infected with lentivirus vectors expressing KPNB1-specific shRNAs or shRNAs + KPNB1 plasmid. B. RT-qPCR results showing KPNB1 expression in U87MG and U251MG cells. Data presented as the mean ± SD of three independent experiments, ***P < 0.001, one-way ANOVA. U87MG and U251MG cells infected with lentivirus vectors expressing KPNB1 specific shRNAs or shRNAs + KPNB1 plasmid after puromycin selection were harvested for colony formation assay (C), Transwell invasion assay (D), and fluorescein isothiocyanate (FITC)/PI flow cytometry (F). Scale bars: 100 μm. Each bar represents the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA. U251MG cells were transfected with the indicated constructs. E. EdU assay of U87MG and U251MG cells infected with lentivirus vectors expressing KPNB1-specific shRNAs or shRNAs + KPNB1 plasmid after puromycin selection. EdU-positive nuclei (red) and 1× DAPI-stained nuclei of all cells (blue) were visualized by fluorescence microscopy. Quantification of the EdU assay on the right. Error bars represent mean ± SD one-way ANOVA, Tukey’s multiple comparisons test; n = 3 independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001. G. After puromycin selection, cells were administered to nude mice by intracranial injection to establish a xenograft model. Representative bioluminescence imaging of a tumor at day 28 is shown. H. Quantitative assessments of tumor growth following implantation. Data expressed as mean ± SD, n = 10 per group. *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA. I. Mice were sacrificed at the ethical endpoint, and survival curves were plotted in Kaplan–Meier graphs, with differences evaluated using the log-rank test (P < 0.001 for control vs. shKPNB1; P < 0.001 for shKPNB1 vs. shKPNB1+ KPNB1)