(A) PC3MM2 cells grown in MEM with 5.5 mM glucose were treated with AEE788 (5.0 μM, with AEE788 readded every 24 hr) for 72 hr. Western blot analysis revealed that pEGFR was completely blocked by AEE788 compared with the control. β-actin served as a loading control (tEGFR, total EGFR).
(B) Seventy-two hours later after cells were cultured in MEM with 5.5 mM glucose, tEGFR and pEGFR levels were both reduced by siRNA treatment compared with the control that was transfected with siRNA vector-expressing scrambled sequences. β-actin served as a loading control.
(C) Compared with the control cells (there was no difference between non-transfected control cells and control siRNA vector transfected cells), AEE788 only caused a decrease in the proportion of cells in S phase (p < 0.01, arrowhead) with no detectable sub-G1 cells (dead cells). However, the EGFR siRNA-treated cells showed a significant (p < 0.01) proportion of sub-G1 cells (arrow).