Fig. 2.

The silencing effect of the siG12D LODER in human pancreatic cancer cells. (A) Panc1 cells were transiently transfected with siG12D, nontargeting scrambled siRNA (si-scr) or mock transfected using Lipofectamine 2000. A relative level of KRAS mRNA was assessed by real-time PCR. HPRT and UBC were used as endogenous controls. The result is shown as an average of three different samples ± SEM. (B) Panc1-Luc cells were incubated with LODERs containing either siGFP or siG12D in the presence of Lipofectamine 2000 or transiently transfected with these siRNAs using Lipofectamine 2000. After 48 h, cells were lysed and the total level of KRAS protein was assessed by Western blot analysis. Shown are representative blots of 48 h of four independent experiments. (C and D) Panc1 cells were left untreated (u/t) or transiently transfected with siG12D, nontargeting scrambled siRNA (si-scr), or mock transfected using Lipofectamine 2000. At 36 h after the transfection, cells were lysed and relative levels of KRAS, P-Erk, and P-Akt proteins were assessed by Western blot analysis. The results were normalized to the level of β-actin. (C) Representative blots of KRAS, P-Erk, P-Akt, and β-actin. (D) The graph shows the average relative protein level as percentage of mock transfected cells ± SEM. Student t test (p) was calculated compared with mock transfected cells. (E and F) Panc1-Luc cells were incubated with LODERs containing either siGFP or siG12D in the presence of Lipofectamine 2000 for 48, 72, 96, or 120 h. Cell viability (E) and cell death (F) were assessed using XTT and LDH tests, respectively. Representative results of six different samples are shown ± SEM, *P < 0.05; **P < 0.01 according to the Student t test.