Figure 4. Knockdown FOXD3 activated EGFR/Ras/Raf/MEK/ERK signal pathway in the human colon cancer cells.

A and B. HCT116 and Caco-2 cells were treated with scrambled siRNA (control) or FOXD3 siRNA for 72h and then collected for Western Blot analysis. Total protein was isolated from HCT116 and Caco-2 cells and analyzed by immunoblotting with the antibodies against EGFR, K-Ras, p-RAF, p-MEK, MEK, p-ERK and ERK. C. HCT116 cells were treated with scrambled siRNA (control) or FOXD3 siRNA 1^# and 2^# for 72h and then collected for Western Blot analysis. D. HCT116 and Caco-2 cells were treated with FOXD3 siRNA with or without EGFR siRNA for 72h. Total protein was isolated from cells and analyzed by immunoblotting with the antibodies against FOXD3, EGFR and K-Ras. E. Half maximal inhibitory concentration (IC50) of ERK inhibitor. Human colon cancer were transfected with scrambled siRNA (control) or FOXD3 siRNA for 72h and then treated with various concentrations of ERK inhibitor for 24 h, and the IC50 values were determined by analyzing the data with GraphPad Prism 3.03. The X axes in each graph is presented as log10 values, and the data are plotted as the mean ± SD. *p<0.05, SiFOXD3 versus control. F. Rescue blanket of blanket FOXD3 siRNA-mediatedeffectsby reintroducing a FOXD3 expression vector in HCT116 and Caco-2 cells. Negative control cells and FOXD3 knockdown cells transfected with or without blanket FOXD3 expression vector were collected for Western Blot analysis G. Cell viability of control cells and FOXD3 knockdown cells transfected with or without FOXD3 expression vector was monitored by MTT assay in HCT116 and Caco-2 cells. Data are expressed as mean ± SD (n = 6).*p<0.05, **p<0.01.