Figure 2

ET-1 induces CXCR4 mRNA and protein expression in 6-10B nasopharyngeal carcinoma cells in a time- and concentration-dependent fashion (A-F). Initially, 6-10B cells were serum-starved for 24 hours and then stimulated with increasing concentrations (0, 0.1, 1, 10, and 100 nM) of ET-1 for 24 hours or with 10 nM ET-1 for the indicated time. Total RNA was extracted and analyzed using real-time PCR for CXCR4 mRNA expression. Primers for GAPDH mRNA were used as a loading control (A, B). Whole-cell lysates were prepared, and CXCR4 levels were analyzed by western blotting (C, D) and flow cytometry (E, F). As an internal control, the membranes were reprobed with a specific anti-GAPDH antibody. CXCR4 induction was maximal in response to 10 nM ET-1 following a 24-hour exposure. Higher concentrations of ET-1 did not induce further CXCR4 expression. CXCR4 expression increased within 1 hour and remained high at 48 hours. siETAR reduced ETAR and CXCR4 protein expression and attenuated the ET-1-induced stimulation of CXCR4 expression in 5-8F cells (G-I). siRNA transfection was performed per the protocol. Thirty-six hours after transfection, the cells were harvested, whole-cell lysates were prepared, and the CXCR4 and/or ETAR levels were analyzed by western blotting. α-tubulin was used as internal control.NC is a negative control with scrambled siRNA. Representative results from one of three independent experiments are shown.