Figure 3. Decreased phosphorylation of 4E-BP1 correlates with increased apoptosis.
(A) HCT116 and (B) DLD-1 cells were plated in 6 well plates at a density of 1.2×105 cells/well. Cells were transfected 12 h later with 100 nM NTC siRNA, 50 nM PIK3CA siRNA + 50 nM NTC siRNA, 50 nM KRAS siRNA + 50 nM NTC siRNA, or 50 nM PIK3CA siRNA + 50 nM KRAS siRNA. RNA was isolated 48 h post transfection and PIK3CA and KRAS mRNA levels measured using qRT-PCR (* p < 0.05 vs. control; RQ = relative quantity). HCT116 and DLD-1 cells were again plated and transfected under the same conditions to evaluate corresponding protein expression. Whole cell lysates were collected at 72 h and analyzed by Western blot (20 to 60 ug total protein per lane). β-actin was used as a loading control.