Figure 5

The CXCL12-CXCR4 axis is required for EGFR-L858R–dependent increases in invasive properties.

(a) CXCR4 in H1299-EGFR-L858R cells was knocked down using a specific siRNA. Cells were harvested 48 hours after transfection and CXCR4 expression was analyzed by real-time RT-PCR (*P < 0.01). si-Scramble, cells transfected with scrambled control siRNA; si-CXCR4, cells transfected with CXCR4-specific siRNA. (b) The invasion ability of H1299-EGFR-L858R cells was determined using a modified Boyden chamber after siRNA-mediated knockdown of CXCR4 (*P < 0.01). (c) The EGFR-L858R mutant enhances CXCL12-CXCR4–mediated cell invasiveness. H1299-EGFR-WT and H1299-EGFR-L858R cells were serum-starved for 24 hours, after which invasion ability was assessed in the absence or presence of CXCL12 (30 ng/ml) (*P < 0.01). (d) H1299-EGFR-L858R cells were pretreated with a CXCR4 neutralizing antibody (12G5), isotype control antibody (IgG control), or CXCR4 antagonist (AMD3100) for 30 minutes. The invasion ability of cancer cells in the presence of CXCL12 (30 ng/ml) was then evaluated using a modified Boyden chamber assay. The results are presented as changes relative to the control expressed as means ± SD of three independent experiments. P-values (two-sided) were determined using Student’s t-test.