FIGURE 1.

CD166 was overexpressed in liver cancer and protected cells from apoptosis. A, representative IHC images of CD166 staining in liver cancer and adjacent normal liver tissues. B, CD166 expression pattern in different cell lines as measured by Western blotting. S, shorter exposure; L, longer exposure. C, phase-contrast images of Bel-7402 cells expressing shRNA against GFP (Control) or CD166 (sh1). Cells were plated at a density of 5,000 cells/well and imaged 6 days later. Scale bar, 60 μm (left). Cells were then quantified, and the data are shown as mean ± S.D. (error bars) from three independent tests (right). p < 0.01 indicates statistic significance. D, Western blots of Bcl-2 in control (infected with GFP-shRNA) and Bel-7402 cells with CD166 knocked down (infected with sh1 and sh2, respectively). E, CD166 knockdown-induced apoptosis as measured by cleaved caspase-3 staining. Cells were plated at a density of 2,000 cells/well and harvested for immunofluorescence 24 h later. Areas indicated by a rectangular box were enlarged to show cleaved caspase-3 localization. Scale bar, 30 μm (top). Cleaved caspase-3-positive cells were then quantified, and the data are shown as mean ± S.D. from three independent tests (bottom). p < 0.01 indicates statistical significance. F, knockdown efficiency of CD166-specific shRNA (sh1) as measured in Bel-7402 and SMMC-7721 cells by Western blotting. G, knockdown of CD166 enhanced doxorubicin-induced apoptosis as measured by caspase-3/7 activity in control (infected by GFP-shRNA) and Bel-7402 cells with CD166 knocked down (infected by CD166-sh1) treated with DMSO or doxorubicin (0.5 μg/ml). p < 0.01 indicates statistic significance. H, in vivo metastasis analysis indicated no difference in the lung before and after overexpression of CD166. 1 × 10⁶ control (transfected with empty plasmid) or Bel-7402 cells with CD166 overexpressed (transfected with CD166-expressing plasmid) were injected into tail veins of 8-week-old nude mice, which were sacrificed 4 weeks later. The mice were then necropsied, and the lungs were removed. I, Western blots of VEGFR1/2 in control (infected with GFP-shRNA) and Bel-7402 cells with CD166 knocked down (infected with CD166-sh1).