Effect of fisetin, 5-FU or their combination on the phosphorylation of mTOR target proteins and constituents of the mTOR signaling complex in PIK3CA-mutant human colon cancer cells. (a) Effect of fisetin, 5-FU or their combination on the phosphorylation of mTOR target proteins in HCT116 cells, and (b) HT29 cells. (c) Effect of fisetin, 5-FU or their combination on the protein expression of constituents of the mTOR signaling complex in HCT116 cells, and (d) HT29 cells. As described in the Materials and Methods, the cells were treated with fisetin (30–90 μM), 5-FU (50 μM) and their combination for 48 hr and then harvested. Total cell lysates were prepared and 40 μg proteins was subjected to SDS-PAGE followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for vinculin.