Fig. 4.

Concurrent inhibition of FAS and HER2 signalings synergistically enhances apoptotic cell death. (A) SK-Br3 cells were treated with 10 μg/ml trastuzumab, 2 μg/ml cerulenin, or a combination of trastuzumab and cerulenin. Seventy-two hours later, quantification of apoptotic cell death was determined by Cell Death ELISA (Roche Molecular Biochemicals) per the manufacturer's instructions. Data are expressed as absorbance by using the formula [A₄₀₅ - A₄₉₀]_TREATED/[A₄₀₅ - A₄₉₀]_UNTREATED. (B) SK-Br3 cells were grown in eight-well chamber slides and treated with 5 μg/ml trastuzumab, 1 μg/ml cerulenin, or a combination of 5 μg/ml trastuzumab plus 1 μg/ml cerulenin. Seventy-two hours later, TUNEL analyses were performed by using the DeadEnd Fluorometric TUNEL System per the manufacturer's protocol. The immunofluorescence photomicrographs (i–iii) of cells undergoing apoptosis (green staining) and the corresponding 4′,6-diamidino-2-phenylindole-counterstained cells are shown. The number in the lower right of each panel represents the percentage of TUNEL-positive cells. (C) (Upper) SK-Br3 cells were grown in eight-well chambers and transfected with 50 nM FAS siRNA, 50 nM HER2 siRNA, or a combination of 50 nM FAS siRNA plus 50 nM HER2 siRNA. Seventy-two hours later, TUNEL analyses were performed by using the DeadEnd Fluorometric TUNEL System. The number in the upper right of each panel represents the percentage of TUNEL-positive cells. (Lower) The amount of p185^HER2 in FAS RNAi- and/or HER2 RNAi-transfected SK-Br3 cells was quantified by flow cytometry with the anti-p185^HER2 Ab-5. The mean fluorescence signal ± SD (n = 3) was quantified by using the Geo Mean fluorescence parameter provided with cellquest software.