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. 2015 Jul 23;290(36):21901–21914. doi: 10.1074/jbc.M115.670976

FIGURE 8.

FIGURE 8.

Inhibition of PAX3 and FOXD3 function leads to a significant decrease in motility, migration, and chemotaxis in melanoma cells, but this inhibition is reversed with the addition of exogenous CXCR4. A and B, dominant-negative PAX3 and FOXD3 proteins reduce cellular motility (A), whereas added CXCR4 restores this loss (B). Cellular scratch assays of cells were transfected with either pcDNA3 (control group), or with FOXD3-DN, PAX3-DN, and/or CXCR4 expression constructs. Graphs are shown as percent scratch area closed, calculated as measurements of the wound area at 6 (A375) or 24 h (mel-624) post-creation of the scratch, divided by the measurements at the start of the experiment and multiplied by 100. Dominant-negative proteins demonstrated decreased motility compared with control groups (graphs in A, A375 p ≤ 0.05, mel-624 p ≤ 0.005). The addition of CXCR4 restored cellular motility (CXCR4 group versus PAX3-DN + FOXD3-DN + CXCR4 groups, p > 0.17 both cell types). C and D, dominant-negative PAX3 and FOXD3 proteins decrease cellular migration and chemotaxis, whereas added CXCR4 restores this loss. Melanoma cells were tested in transwell assays with FBS and SDF1 added to the lower chamber as a chemoattractant (shown schematically in C) that were transfected with either pcDNA3 (control groups), or with vectors expressing CXCR4, PAX3-DN and FOXD3-DN, or both (D). For each group, six fields of cells for each experiment was counted and averaged. The addition of CXCR4 increased migration significantly (66.72 ± 5.45 cells/field (pcDNA3) to 107.17 ± 32.19 (CXCR4) for A375, and 69.33 ± 7.54 (pcDNA3) to 115.67 ± 15.09 for mel-624, p ≤ 0.05 both cell lines). The presence of both dominant-negative proteins significantly inhibited migration (42.72 ± 12.45 for A375, 41.94 ± 12.70 for mel-624, p ≤ 0.05 both cell lines). Adding CXCR4 to cells transfected with both dominant-negative proteins restored cellular migration (84.50 ± 24.90 for A375, 104.28 ± 10.22 for mel-624, p ≥ 0.19 when comparing CXCR4 groups to PAX3-DN + FOXD3-DN + CXCR4 groups, both cell lines).