FIGURE 5.

Effects of metformin and Sp knockdown on EGFR expression and Ras activation. A, RNA interference with siRNA against EGFR (siEGFR) decreased cell proliferation. Cells were transfected with siRNA against lamin or EGFR, and cells were counted after 48 h as described under “Experimental Procedures.” B, metformin (Met) decreased EGFR expression. Panc28 and L3.6pL cells were treated with 5, 10, and 20 mm metformin, and expression of EGFR was analyzed by Western blot analysis. C, knockdown of Sp proteins decreased expression of EGFR. Cells were transfected with iLamin, iSp1, iSp3, or iSp4, or a mixture of iSp1, iSp3, and iSp4, and expression of EGFR was analyzed by Western blot analysis. iCtrl, control knockdown. D and E, metformin or knockdown of Sp proteins and EGFR decreased levels of active Ras (Ras-GTP). Panc28 and L3.6pL cells were treated with or without metformin or transfected with small inhibitory RNA for lamin or Sp (a mixture of iSp1, iSp3, and iSp4) (D) or knockdown of EGFR (iEGFR) (E). Levels of Ras-GTP were determined using active Ras detection assay. GTPγS and GDP act as positive and negative controls, respectively. Activated Ras was quantified and normalized to total Ras levels. Results are expressed as the mean ± S.E. for at least three separate determinations, and a significant (p < 0.05) growth inhibition (A), decrease in EGFR expression (C), and decreased Ras activity (D) are indicated (*).