The impact of MEK1/2-ERK1/2 and PI3K-Akt inhibition on EGF-induced effects in HT-29 cells.

<p>(<b>A</b>) HT-29 cells were grown and treated with EGF for 5, 15, 30 and 60 min, after which the total cell lysates were harvested and analyzed by immunoblotting for p-ERK1/2, ERK1/2, p-Akt, and Akt. (<b>B</b>) Cells were grown and pretreated for 1 h with the indicated inhibitors before incubation with EGF for 48 h. Total cell lysates were harvested and analyzed by immunoblotting for claudin-3. α-Tubulin was used as a loading control. The numbers represent the ratio of the optical density of treated to untreated cells normalized by total protein or α-tubulin. Representative images of wound healing (<b>C</b>), anchorage-dependent colony formation (<b>D</b>) and anchorage-independent colony formation (<b>E</b>) assays. The cells were pretreated with the inhibitors as indicated, and the assays were performed as described in the Materials and Methods. For the anchorage-dependent assay, the bar graph shows the ratio of the fold increase in foci formation of treated to untreated cells (control). For the anchorage-independent assay, the bar graphs show the ratio of the fold increase in colony formation of treated to untreated cells (control). Bar: 200 µm. Error bars indicate the means ± SEM (n = 3); **p<0.01 as determined by an ANOVA.