Figure 3

Knockdown of Crabp2/CRABP2 suppresses migration, invasion, anoikis resistance, and in vivo metastasis. (a) Western blot analysis of Crabp2/CRABP2 and β-actin (ACTB, as the loading control) in C10F4 or H1650 cells expressing control-shRNA (shCon) versus Crabp2-shRNA (shCrabp2) (left), control-siRNA (siCon) versus Crabp2/CRABP2-siRNA (siCrabp2/siCRABP2) (middle and right). (b,d) Migration assay of C10F4 (b) or H1650 (d) cells expressing control-siRNA versus Crabp2/CRABP2-siRNA for 12 hours. Cells migrated into the lower compartment of Boyden chamber were photographed (left) and quantified (right). (c,e) Matrigel cell invasion assay of C10F4 (c) or H1650 (e) cells expressing control-siRNA versus Crabp2/CRABP2-siRNA for 15 (C10F4) or 12 (H1650) hours. Cells invaded through the matrigel were photographed (left) and quantified (right). (f) Anoikis assay of C10F4 cells expressing control-siRNA versus Crabp2-siRNA (left), or control-shRNA versus Crabp2-shRNA (right). Cells were plated onto a 96-well anchorage-resistant plate at a density of 10,000 cells/well. Twenty-four hours later, viability of cells was assessed by MTS assay. The p values were determined using Student’s t test. (g) Anoikis assay of H1650 cells expressing control-siRNA versus CRABP2-siRNA. Cells were plated onto a 96-well anchorage-resistant plate at a density of 10,000 cells/well. Twenty-four hours later, viability of cells was assessed by MTS assay. The p value was determined using Student’s t test. (h) Metastasis of GFP⁺ C10F4 cells expressing control-shRNA (n = 3) versus Crabp2-shRNA (n = 3). One million cells were injected into tail veins of each BALB/c mouse. Twelve days later, mouse lungs and livers were harvested and analyzed using H&E staining (left), and metastatic cells at lung were analyzed using real-time RT-PCR of GFP RNAs (right). Tumor regions were indicated by T or yellow triangle. The p value was determined using Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.