Semaphorin 3A Increases FAK Phosphorylation at Focal Adhesions to Modulate MDA-MB-231 Cell Migration and Spreading on Different Substratum Concentrations

Figure 6

Inhibition of ROCK blocks the Sema3A-mediated effects on cell migration, spreading, and pFAK³⁹⁷ levels at focal adhesions. (a) Representative phase-contrast images from a scratch assay of cells cultured on substrata coated with 10 μg/mL collagen in the presence of 100 μg/mL IgG1 Fc control or Sema3A with and without 10 μM Y-27632. Scale bar = 100 μm. (b) Y-27632 blocked the Sema3A-mediated effects on all coating concentrations of collagen as measured using % area closure. Data are presented as average % area closure ± SEM from four independent experiments. (c) Average cell area of cells cultured on substrata coated with low, intermediate, and high concentrations of collagen. Cells were treated with IgG1 Fc control or Sema3A in the absence or presence of Y-27632. Y-27632 blocked the effects of Sema3A on all coating concentrations of collagen. Graphed data represent the mean ± SEM from at least 120 cells for each condition. ((d) and (e)) The effects of Y-27632 on control- and Sema3A-treated cells were assessed using cell circularity (d) and aspect ratio (e). While Sema3A alone decreased circularity and increased aspect ratio on substrata coated with low and intermediate concentrations of collagen, Y-27632 alone produced a similar effect. Data are presented as mean ± SEM from a minimum of 120 cells for each condition. (f) Y-27632 inhibited pFAK³⁹⁷ staining levels (green) at focal adhesions in both IgG1 Fc control- and Sema3A-treated conditions. Cells were counterstained using TRITC-phalloidin (red). Scale bar = 10 μm. ((g) and (h)) The average total surface area of pFAK³⁹⁷ (g) and relative pFAK³⁹⁷ normalized to total cell area (h) for control- and Sema3A-treated cells exposed to substrata coated with low, intermediate, and high concentrations of collagen in the presence or absence of Y-27632. Graphed data represent the mean ± SEM from at least 50 cells for each condition. , , and indicate statistical significance relative to IgG1 Fc control for all graphed data; two-sample -test.

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