Fig. 2

Trastuzumab causes cell cycle arrest and cell death in trastuzumab-hypersensitive cells. Trastuzumab-resistant and trastuzumab-hypersensitive models maintain HER2 levels (A-E). HER2 surface levels were analyzed by flow cytometry (A), immunofluorescence (B) and cell surface immunoprecipitation (E) using trastuzumab 10 nM as primary antibody in all cases. Total HER2 levels were determined by immunofluorescence (C) or western blot (D). Levels of total (D) and cell surface tyrosine-phosphorylated HER2 were determined by western blot. For immunofluorescence, cells were fixed and stained for HER2 using trastuzumab (green) (B) or an anti-HER2 antibody (red) (C) and DNA (blue). Scale bar, 10 μm. F Evaluation of S phase entry upon trastuzumab treatment. Cells were treated with trastuzumab for 5 days, received a 10 μM pulse of BrdU and BrdU incorporation was determined by flow cytometry. Data is represented as the mean ± SD, normalized to untreated controls of two independent experiments. G Effect of trastuzumab on cell death. Cells were treated with trastuzumab for 6 days and stained with annexin V-FITC and propidium iodide, followed by flow cytometry analysis. The graph shows mean ± SD of viable and non-viable cells from two independent experiments. I Trastuzumab treatment induces caspase 3 activation in BTSH cells. Cells were treated with trastuzumab for 6 days and caspase 3 activity was determined by fluorometric techniques using a caspase 3 specific substrate. H Analysis of proapoptotic and cell cycle progression proteins after trastuzumab treatment. Cells were treated with trastuzumab for 6 days and the amount of the indicated proteins was assessed by western blot. cCaspase stands for cleavage caspase. Calnexin was used as a loading control. C: control; T: trastuzumab; IP: immunoprecipitation; WB: western blot