Figure 7. AEG-1 regulates CXCR4 expression to mediate CXCR4/CXCL12-dependent orientation chemotaxis.

(A) To assess the role of the CXCR4/CXCL12 axis in orientation chemotaxis, HCC cells were seeded onto plates that were pre-coated with 2% again and incubated for 24h and then lysates were examined by Western blotting for CXCR4. GAPDH was tested as a loading control. Results from a representative experiment (left) and the mean±SD epressionexpression values for three independent experiments (right) are shown (*P<0.05 vs the corresponding control cell). (B) CXCL12 secretion in HPMECs, HHSECs and HUVECs was assessed by ELISA. Results represent the mean±SD of 3 independent experiments (*P<0.05) (C) Cell proliferation of AEG-1-over-expressing SMMC-7721 cells treated with or without AMD3100 (10 µM) was assessed by MTT over a timecourse of AMD3100 addition (*P>0.05). (D) AEG-1-over-expressing SMMC-7221 cells were cultured in suspension for 24 h and then pretreated with 0 to 20 µM or without AMD3100 from 0 to 20 (10 µM) for 2 h. Cells were assessed for chemotaxis toward HPMEC serum-free endothelial cell medium (*P<0.05) supernatant. Results from a representative experiment are shown (left) and the mean + SD of five different fields of three independent experiments are quantified (right; mean±SD; *P<0.05). (E) AEG-1-over-expressing SMMC-7221 cells were cultured in suspension for 24 h and then pretreated with or without AMD3100 (10 µM) for 2 h. Cells were assessed for chemotaxis toward HPMEC supernatant as above. Results from a representative experiment (left; ×400, HE), and the quantification of five different fields of three independent experiments (right) are shown (mean±SD; *P<0.05).