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. 2016 May 24;291(30):15540–15550. doi: 10.1074/jbc.M115.712976

FIGURE 6.

FIGURE 6.

Inhibition of Src activity abrogates the phosphorylation of FAK-Tyr925 and cell migration induced by CD133. A and B, SW620 cells expressing shLacz or shCD133, cultured in serum starvation condition, were treated with 10 μm PP2 or DMSO for 24 h. The level of Src phosphorylation (pSrc-Tyr416), FAK phosphorylation (pFAK-Tyr397, pFAK-Tyr576/577, and pFAK-Tyr925), total Src, FAK, and CD133 were detected by Western blotting analysis. C, HEK293T cells ectopically expressing CD133 were treated with 10 μm PP2 or DMSO for 24 h. Then the levels of pFAK-Tyr925, total FAK, and CD133 were determined by Western blotting analysis. D–G, SW620 cells expressing shLacz or shCD133 were treated with 10 μm PP2 or DMSO. D and E, representative images of the wound healing assay of SW620 cells with the indicated treatments. Relative distance of cell migration was calculated and is presented as the mean ± S.D. (error bars) (n = 6). Statistical analysis was performed by Student's t test (**, p < 0.01; ***, p < 0.001; ns, not significant). F and G, representative images of the Transwell assay of SW620 cells with the indicated treatments. The number of migratory cells was calculated and is presented as the mean ± S.D. (error bars) (n = 3) Statistical analysis was performed by Student's t test (***, p < 0.001; ns, not significant). H, SW620 cells expressing shLacz or shCD133, cultured in complete medium, were treated with 10 μm PP2 or DMSO for 24 h. Cell proliferative ability was analyzed by Cell Counting Kit 8 in a 96-well plate. A450 was determine, and results are presented as the mean ± S.D. (error bars) (n = 12). Statistical analysis was performed by Student's t test (***, p < 0.001). I and J, HEK293T cells ectopically expressing CD133 were treated with 10 μm PP2 or DMSO for 24 h. I, representative image of the wound healing assay of HEK293T cells with the indicated treatments. J, relative area of cell migration in I was calculated and is presented as the mean ± S.D. (error bars) (n = 6). Statistical analysis was performed by Student's t test (***, p < 0.001). K, HEK293T cells ectopically expressing CD133 were treated with 10 μm PP1, 10 μm PP3, or DMSO for 24 h. Relative area of cell migration was calculated and is presented as the mean ± S.D. (error bars) (n = 6). Statistical analysis was performed by Student's t test (***, p < 0.001; ns, not significant). IB, immunoblotting.