Figure 3. HER4 silencing decreases cell viability and enhances trastuzumab responses.

(A) SKBR3 and BT474 cells were transfected with HER4 siRNA₁₀ and siRNA₁₁, with and without 40μg/ml trastuzumab treatment before the mRNA levels were measured by RT-PCR. The scr (scramble) siRNA was used to normalize for transfection efficiency. Expression of HER4 is relative to the untreated scramble control and is normalised to β-actin. (B) Representative western blots of the indicated proteins from HER4 siRNA experiments in (A) are shown. (C) Cell count experiments were performed in SKBR3 and BT474 cells after 72-hour transfection as in (A) and the relative cell counts (normalized to the scr control) is shown in the graph. Means ± SD of three independent experiments is shown (**p<0.01 ***p<0.001). (D) The effect of HER4 siRNAs in SKBR3 and BT474 cells was further investigated in colony formation experiments after 7 days post transfection as in (A). Representative colony pictures are shown on the upper panels and the relative colony counts (means ± SD) are shown on the bottom panels. (E) SKBR3 and BT474 cells transfected with transient HER4 siRNA (with and without trastuzumab) for 72 hours were stained for Annexin V and the percentage of positive cells was assessed by FACS analysis. Means ± SD from three independent experiments are shown in the graph (*p<0.05, ***p<0.001).