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. 2009 May 6;101(9):663–677. doi: 10.1093/jnci/djp063

Figure 7.

Figure 7

Clusterin and an experimental model of metastasis in mice bearing xenotransplanted human neuroblastoma. A) Western blot analysis of clusterin expression in human neuroblastoma cell lines LA-N-5 and HTLA230. Cells were lysed and subjected to Western blot analysis with a clusterin antibody. Positions of the human 60-kDa clusterin precursor (pClu) and the mature 36-kDa secreted (sClu) clusterin are shown. Actin was used as the loading control. B) Quantification of neuroblastoma cells recovered from BM or liver of mice after 5 weeks that had been injected with LA-N-5 cells transduced with empty or clusterin MIGR1 (Clusterin) vectors (n = 4 mice per group). Tumor cells which were positive for green fluorescent protein expression from the MIGR1 vector were counted by flow cytometry. The statistical significance between the clusterin-transduced and untransduced groups was assessed with Student t test. C) Western blot analysis of clusterin expression in HTLA clones. HTLA cells were stably transfected with control vector (Empty), a plasmid containing short hairpin RNA control sequence 1 (termed HTLA-16 cells), or a plasmid containing clusterin short hairpin RNA sequence 2 (termed HTLA-19b cells), and then subjected to Western blot analysis for clusterin with a clusterin antibody. Actin was used as the loading control. D) Luciferase assays and the activity of NF-κB in HTLA cells with (HTLA-16 cells) or without (HTLA-19b cells) clusterin expression. A NF-κB luciferase reporter construct was transiently transfected into HTLA-16 and -19b cells. Firefly luciferase activity was measured and normalized to that of Renilla luciferase. Statistical significance was assessed with the two-sample Welch t test (n = 3 independent experiments). Error bars = 95% confidence intervals. E) Quantification of neuroblastoma cells recovered from BM of mice injected with HTLA clones HTLA-16 or -19b (n = 8 mice per group). Grey diamonds = HTLA-16 cells; black diamonds = HTLA-19b cells. Statistical significance was assessed with Student t test. All statistical tests were two-sided. NF-κB = nuclear factor κB; BM = bone marrow.