Figure 2.

EGFR knockdown does not affect lapatinib sensitivity of HER2-overexpressing cells. A, EGF stimulation leads to EGFR activation. BT-474 and SK-BR-3 cells were cultured in serum-free medium for 24 h before EGF stimulation. Cells were exposed to EGF in cultured medium at a concentration of 100 ng/mL for 5 min. Western blot analysis was done to detect EGFR and phosphorylated EGFR, Akt, and extracellular signal-regulated kinase 1/2. β-actin was used as a loading control. B, BT-474 and SK-BR-3 cells were transfected with control siRNA or EGFR siRNA for 48 h. Western blot analysis was done to determine the expression level of EGFR after siRNA knockdown. β-actin was used as a loading control. The relative intensity of EGFR was quantified with the NIH Image program. Ratios were calculated to compare the densities of EGFR expression in EGFR siRNA –transfected cells versus control siRNA–treated cells. C, at 48 h after siRNA knockdown, the cells were treated with the indicated concentrations of lapatinib for another 72 h. Growth-inhibitory effects of lapatinib on BT-474 and SK-BR-3 cells were studied by WST-1 assay. Viability of EGFR knockdown cells was compared with that of control siRNA knockdown cells (P > 0.05). Each experiment was repeated thrice independently.