Figure 6.

Clusterin and in vitro invasion of neuroblastoma. A) WB analysis of clusterin expression in neuroblastoma cells after transfection with siRNA against clusterin. Cell lysates were collected 24 hours after transfection of control sequence 1 (Seq 1) or clusterin sequence 2 (Seq 2) and then subjected to WB analysis with antibodies against clusterin to detect precursor (pClu) and secreted (sClu) clusterin. Expression of housekeeping genes, as a loading control, was monitored by WB with actin (HTLA cells) or keratin antibodies (IMR32 and SH-SY5Y cells). B) In vitro invasion assay with cells transfected with siRNAs against clusterin. The numbers of IMR32, HTLA230, and SH-SY5Y cells that migrated to the bottom chamber after mock transfection (wt), transfection of control sequence 1 (Seq1), or clusterin sequence 2 (Seq2) siRNAs are indicated. C) In vitro invasion assay with cells transfected with other siRNAs against clusterin. Left) WB analysis with a clusterin antibody of IMR-32 cells transfected with clusterin (Clu2) or control (Ctr) siRNAs. Middle) Crystal violet staining of invading IMR32 cells. Scale bars = 0.05 mm. Right) In vitro invasion assay results with cells transfected with the control (Ctr) or clusterin (Clu2) siRNAs. Statistical significance was calculated with a Student t test (n = number of independent assays). Error bars = 95% confidence intervals. All statistical tests were two-sided. siRNA = small interfering RNA; WB = Western blot.